KR20200133365A - GIPR antibody and fusion protein thereof with GLP-1, and pharmaceutical composition and application thereof - Google Patents

Abstract

Provided herein are a gastric inhibitory polypeptide receptor (GIPR) antibody and a fusion protein of a glucagon-like peptide-1 (GLP-1) therewith, and a pharmaceutical composition thereof. In addition, the present application uses a GIPR antibody and a fusion protein of GLP-1 therewith to treat, prevent, or ameliorate one or more symptoms of non-alcoholic fatty liver disease, non-alcoholic steatohepatitis, type 2 diabetes or obesity. A method is provided.

Description

GIPR antibody and fusion protein thereof with GLP-1, and pharmaceutical composition and application thereof

Provided herein are a gastric inhibitory polypeptide receptor (GIPR) antibody and a fusion protein of a glucagon-like peptide-1 (GLP-1) therewith, and a pharmaceutical composition thereof. In addition, the present application uses a GIPR antibody and a fusion protein of GLP-1 therewith to treat, prevent, or ameliorate one or more symptoms of non-alcoholic fatty liver disease, non-alcoholic steatohepatitis, type 2 diabetes or obesity. A method is provided.

Intestinal gastric inhibitory polypeptide (GIP) is a polypeptide hormone secreted by intestinal K cells after feeding, and includes two isoforms of a 42 amino acid peptide and a 30 amino acid peptide. GIP is involved in the physiological action of insulin secretion by activating the gastric inhibitory polypeptide receptor (GIPR) on the surface of pancreatic β cells (Tseng et al. , 1996, J. Clin. Invest. 98:2440-2445). ; Ravn et al. , 2013, J. Biol. Chem. 288:19760-72). Because the classical biological function of GIP is similar to GLP-1, these peptide hormones are collectively referred to as incretines. GIPR is widely distributed in many tissues, including pancreas, bone, heart, stomach, intestine and adipose tissue (Peter et al. , 2013, J. Biol. Chem. 288:19760-72), and such a variety of The distribution suggests that the GIP/GIPR pathway has a biological function beyond glycemic control. Experimental evidence shows that the GIP/GIPR signaling pathway is at least closely related to lipid metabolism in these tissues (Yip and Wolfe, 2000, Life Sci. 66:91-103). Experimental data also show an increase in the concentration of circulating GIP in obese or diabetic patients (Creutzfeldt et al. , 1978, Diabetologia 14:15-24); Flatt et al. , 1984, J. Endocrinol . 101:.... 249-256; literature [Salera et al, 1982, J. Clin Endocrinol Metab 55:.... 329-336]; literature [VilsbØll et al, 2003, J. Clin Endocrinol Metab 88 :2706-2713]). After blocking the GIPR signal with a GIPR inhibitor, significant weight loss, decreased insulin resistance and even reversal of type 2 diabetes were observed in obese mice induced by a high fat diet (Ravn et al. , 2013, J. Biol). Chem. 288:19760-72]).

Long-acting glucagon-like peptide-1 analogues (GLP-1 analogues) are the next generation type 2 diabetes drugs (Tomlinson et al. , 2015, Expert Opin. Investig. Drugs 25:1744-7658; Gallwitz, 2015, Eur. Endocr . 11:21-25]). Long-lasting GLP-1 drugs are also being studied in clinical trials for the treatment of non-alcoholic fatty liver disease (NAFLD). Studies show that long-acting GLP-1 drugs significantly affect liver tissue morphology, decrease alanine aminotransferase/glutathione aminotransferase ratio, and liver fat content in NAFLD patients (Samson et al. , 2013, J. Diabetes Complications 27:401-6; Portillo-Sanchez and Cusi, 2016, Clin. Diabetes Endocrinol . 2:9).

When a GLP-1 drug and a GIPR inhibitor can be used together, including a combination of the two, e.g. as a fusion protein, this combination lowers blood sugar while reducing excess fat accumulation (obesity) while improving insulin resistance. It can achieve an effect that interferes with metabolism. In this regard, the GLP-1 moiety can be used to improve glucose tolerance, reduce appetite, lower blood sugar, and lose weight; The GIPR antibody portion can be used to reduce the further accumulation of fat and improve liver function. The fat-reducing effect of GIPR antibody and the weight-loss effect of GLP-1 can be used synergistically to treat non-alcoholic fatty liver disease/non-alcoholic steatohepatitis. The present disclosure provides fusion protein drugs that will benefit patients with one or more of nonalcoholic fatty liver disease/nonalcoholic steatohepatitis, type 2 diabetes and obesity.

Provided herein are antibodies that specifically bind to GIPR, wherein the antibody is an inhibitor of GIPR.

Also provided herein is an antibody that specifically binds to GIPR, comprising a sequence of 1, 2, 3, 4, 5 or 6 amino acids, wherein each amino acid sequence is from the amino acid sequence listed below. Are independently selected:

a. Light chain CDR1 amino acid sequence: SEQ ID NO: 1, SEQ ID NO: 4, SEQ ID NO: 7, SEQ ID NO: 10, SEQ ID NO: 13, and SEQ ID NO: 15;

b. Light chain CDR2 amino acid sequence: SEQ ID NO: 2, SEQ ID NO: 5, SEQ ID NO: 8, SEQ ID NO: 11, and SEQ ID NO: 16;

c. Light chain CDR3 amino acid sequence: SEQ ID NO: 3, SEQ ID NO: 6, SEQ ID NO: 9, SEQ ID NO: 12, SEQ ID NO: 14, and SEQ ID NO: 17;

d. Heavy chain CDR1 amino acid sequence: SEQ ID NO: 18, SEQ ID NO: 23, and SEQ ID NO: 26;

e. Heavy chain CDR2 amino acid sequence: SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 24, SEQ ID NO: 27, and SEQ ID NO: 29;

f. Heavy chain CDR3 amino acid sequence: SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 25, SEQ ID NO: 28, and SEQ ID NO: 30.

Provided herein is an antibody that specifically binds to GIPR, and a GLP-1 fusion protein comprising 1, 2, 3, 4, 5, 6, 7 or 8 GLP-1 fragments, ; The fusion protein connects the carboxy terminus of the GLP-1 fragment with the amino terminus of the light or heavy chain of the GIPR antibody, or connects the amino terminus of the GLP-1 fragment with the carboxy terminus of the light chain of the GIPR antibody.

Provided herein is a GLP-1 fusion protein comprising a GIPR antibody and two GLP-1 fragments; The fusion protein is N'-GLP-1-Linker-R-C', which is a link between the carboxy terminus of the GLP-1 fragment and the amino terminus of the light chain of the GIPR antibody; N'-GLP-1-Linker-R-C', which is the linking of the carboxy terminal of the GLP-1 fragment to the amino terminal of the heavy chain of the GIPR antibody; Where N'represents the amino terminal of the fusion protein polypeptide chain, C'represents the carboxy terminal of the fusion protein polypeptide chain, GLP-1 represents the GLP-1 fragment, and R represents the light or heavy amino acid of the GIPR antibody. Sequence, and Linker represents a peptide linker.

Provided herein are polynucleotides encoding the GIPR antibodies described herein.

Provided herein are polynucleotides encoding the fusion protein of GIPR antibody and GLP-1 described herein.

Provided herein are vectors comprising polynucleotides encoding the GIPR antibodies described herein.

Provided herein is a vector comprising a polynucleotide encoding a fusion protein of GIPR antibody and GLP-1 described herein.

Provided herein are host cells comprising the vectors described herein.

Provided herein are pharmaceutical compositions comprising the GIPR antibodies described herein and a pharmaceutically acceptable carrier.

Provided herein is a pharmaceutical composition comprising a fusion protein of a GIPR antibody and GLP-1 described herein and a pharmaceutically acceptable carrier. Further provided herein is the use of the GIPR antibodies described herein in the manufacture of a medicament for the treatment, prevention or amelioration of nonalcoholic steatohepatitis diseases.

Provided herein is the use of a fusion protein of a GIPR antibody and GLP-1 described herein in the manufacture of a medicament for treating, preventing, or ameliorating non-alcoholic fatty liver disease.

Provided herein is the use of a GIPR antibody described herein in the manufacture of a medicament for treating, preventing or ameliorating type 2 diabetes.

Provided herein is the use of a fusion protein of a GIPR antibody and GLP-1 described herein in the manufacture of a medicament for treating, preventing or ameliorating type 2 diabetes.

Provided herein is the use of the GIPR antibodies described herein in the manufacture of a medicament for reducing body weight or treating, preventing or ameliorating obesity and obesity-related diseases.

Provided herein is the use of a fusion protein of a GIPR antibody and GLP-1 described herein in the manufacture of a medicament for reducing body weight or treating, preventing or ameliorating obesity and obesity-related diseases.

Provided herein is the use of the GIPR antibodies described herein in the manufacture of a medicament for the simultaneous treatment of two or more of nonalcoholic fatty liver disease, obesity, or type 2 diabetes.

Provided herein is the use of a fusion protein of a GIPR antibody and GLP-1 described herein in the manufacture of a medicament for the simultaneous treatment of two or more diseases of nonalcoholic fatty liver disease, obesity, or type 2 diabetes.

Provided herein is a method of treating, preventing, or ameliorating one or more symptoms of nonalcoholic steatohepatitis comprising providing to a subject a therapeutically effective dose of a GIPR antibody described herein.

Provided herein is a method of treating, preventing, or ameliorating one or more symptoms of nonalcoholic steatohepatitis comprising providing to a subject a therapeutically effective dose of a fusion protein of a GIPR antibody described herein and GLP-1. .

Provided herein is a method of treating, preventing, or ameliorating one or more symptoms of type 2 diabetes, comprising providing to the subject a therapeutically effective dose of a GIPR antibody described herein.

Provided herein is a method of treating, preventing, or ameliorating one or more symptoms of type 2 diabetes comprising the step of providing to a subject a therapeutically effective dose of a fusion protein of a GIPR antibody described herein and GLP-1. .

Provided herein is a method of treating, preventing, or ameliorating one or more symptoms of obesity, comprising providing to a subject a therapeutically effective dose of a GIPR antibody described herein.

Provided herein are methods of treating, preventing, or ameliorating one or more symptoms of obesity comprising providing to a subject a therapeutically effective dose of a fusion protein of a GIPR antibody described herein and GLP-1.

1 shows the results of a FACS test of specific binding of a recombinantly expressed hGIPR antibody, L10H8 (including SEQ ID NO: 70 and SEQ ID NO: 79) to hGIPR. The gray peak and the dotted peak are negative control, the gray peak represents the background peak of blank cells CHO-DHFR-, the dotted peak represents the negative binding peak of L10H8 to blank cells CHO-DHFR-, solid peak is The specific binding peak of L10H8 to CHO-DHFR-hGIPR is shown.
Figure 2 shows the hGIPR antibody L7H6 (SEQ ID NO: 67 and SEQ ID NO:) antagonizing GIP activation of the hGIPR signaling pathway, as determined by direct cAMP assay (IC ₅₀ = 7.6 nM, R ² = 0.99). 77).
3 is a GIPR antibody/GLP-1 fusion protein GLP-1-Linker- that antagonizes GIP activation of the hGIPR signaling pathway, as determined by direct cAMP assay (IC ₅₀ = 14.9 nM, R ² = 0.99). Inhibition curves of L7H6 (including SEQ ID NO: 67, SEQ ID NO: 77, SEQ ID NO: 106, SEQ ID NO: 111) are shown.
Figure 4 is a GIPR antibody / GLP-1 fusion protein GLP-1-Linker-L7H6 activating the hGLP-1R signaling pathway, as determined by reporter gene assay (EC ₅₀ = 0.04 nM, R ² = 0.99). The activation curve of the reporter gene experiment to test is shown.
Figure 5 shows the time curve of the rate of body weight change in various groups of C57BL/6 obese mice induced by a high fat diet during the efficacy test period.

Justice

Unless otherwise defined herein, scientific and technical terms will have the meaning understood by one of ordinary skill in the art. In general, nomenclature and techniques related to pharmacology, biology, biochemistry, cell and tissue culture, biology, molecular biology, immunology, microbiology, genetics and protein nucleic acid chemistry, as well as hybridization are well known in the field of the present invention and are common in the field. Is used as

Standard single-letter or three-letter abbreviations are used in the present invention to indicate polynucleotide and polypeptide sequences. When designating a polypeptide sequence, the first amino acid residue (N') with an amino group is on the far left, and the last amino acid residue (C') with a carboxyl group is on the far right, for example, in the present invention. Related GLP-1 fragment sequences are as follows: SEQ ID NO: 105, SEQ ID NO: 106, SEQ ID NO: 107, SEQ ID NO: 108, and SEQ ID NO: 109. Reverse polypeptide sequence is amino acid native It refers to a polypeptide sequence arranged in the reverse order for that of, for example, the reverse GLP-1 fragment sequence converted from the GLP-1 fragment sequence is as follows: SEQ ID NO: 119, SEQ ID NO: 120, SEQ ID NO: 121, SEQ ID NO: 122, and SEQ ID NO: 123. The 5'end of the upstream chain of single-stranded and double-stranded nucleic acid sequences is on the left, and its 3'end is on the right. A specific portion of the polypeptide may be represented by an amino acid residue number, such as amino acids 80 to 130, or may be represented by an actual residue of the site, such as Lys80 to Lys130. A particular polypeptide or polynucleotide sequence can also be described by explaining the difference from the reference sequence.

The terms "peptide", "polypeptide", and "protein" refer to molecules containing two or more amino acids interconnected by peptide bonds. These terms include, for example, natural and artificial proteins, and peptide analogs of protein sequences (such as mutant proteins, variants and fusion proteins), and proteins that have been modified post-transcriptional or otherwise covalently or non-covalently modified. do. The peptide, polypeptide, or protein can be a monomer or a polymer.

The term "polypeptide fragment" refers to a polypeptide that has the amino terminus and/or carboxyl terminus missing from the corresponding full-length protein. For example, the length of fragments is at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 50, 70, It may be 80, 90, 100, 150, or 200 amino acids. Fragment length is, for example, up to 1000, 750, 500, 250, 200, 175, 150, 125, 100, 90, 80, 70, 60, 50 , 40, 30, 20, 15, 14, 13, 12, 11, or 10 amino acids. Fragments may further comprise one or more additional amino acids at one or both ends, such as amino acid sequences from various natural proteins (e.g., Fc or leucine zipper domains) or artificial amino acid sequences (e.g., artificial joint sequences). It may contain.

In the present invention, the peptides are used for any reason and by any means, for example, (1) reducing proteolytic sensitivity, (2) reducing oxidative sensitivity, and (3) increasing affinity to form protein complexes. Altering, (4) altering binding affinity, and (5) imparting or altering other physicochemical or functional properties. Analogs contain mutant proteins of the polypeptide. For example, single or multiple amino acid substitutions (eg, conservative amino acid substitutions) can be performed in the native sequence (eg, outside the domain of the polypeptide forming intermolecular contact). A "conservative amino acid substitution" is a substitution that does not significantly alter the structural characteristics of the parent sequence (eg, substitution of an amino acid must not damage the helix present in the parent sequence or its property or function in the parent sequence. Should not interfere with other secondary structure types required to provide a).

A “mutant” of a polypeptide has an amino acid sequence comprising insertions, deletions, and/or replacements of one or more residues in the amino acid sequence compared to another polypeptide sequence. In the present invention, the variant includes a fusion protein.

A “derivative” of a polypeptide is a polypeptide that has been chemically modified due to binding, phosphorylation, and glycosylation to other chemical components such as, for example, polyethylene glycol, albumin (such as human serum albumin).

Unless otherwise stated, the term “antibody” includes antibodies having two full-length heavy chains and two full-length light chains, as well as derivatives, variants, fragments, and mutated proteins thereof, examples are listed below.

The term "antibody" is a protein containing an antigen-binding portion and, optionally, a scaffold or framework portion that allows the antigen-binding portion to assume a conformation that facilitates binding of the antibody to the antigen. Examples of antibodies include complete antibodies, antibody fragments (such as the antigen-binding portion of an antibody), antibody derivatives, and antibody analogs. For example, the antibody may contain an alternative protein scaffold or artificial scaffold having an grafted CDR or derivative of the CDR. Scaffolds include, but are not limited to, antibody-derived scaffolds to be introduced, such as those that stabilize the three-dimensional structure of antibodies, and fully synthetic scaffolds for biocompatible polymers. See, eg, Korndorfer et al. , 2003, Proteins 53:121-129]; Roque et al. , 2004, Biotechnol. Prog. 20:639-654. In addition, the antibody may be a mock peptide antibody ("PAMs") or a scaffold containing a mock antibody, wherein a fibrin ligand is used as the scaffold.

Antibodies can have a native immunoglobulin-like structure. "Immunoglobulin" is a tetrameric molecule. In natural immunoglobulins, each tetramer consists of two identical pairs of polypeptide chains, each pair having a "light" chain (approximately 25k Da) and a "heavy" chain (approximately 50 kDa to 70 kDa). The amino terminus of each chain contains a variable domain of about 100 to 110 amino acids primarily involved in antigen recognition. The carboxyl terminus of each chain determines the constant region primarily associated with the effect of the effector. The human antibody light chain is divided into a κ light chain and a λ light chain. The heavy chain was divided into μ, δ, α, or ε, and the same type of antigen was determined, such as IgM, IgD, IgG, IgA, and IgE. In the light and heavy chains, the variable and constant regions are linked by a "J" region of about 12 or more amino acids, and the heavy chain also comprises a "D" region of about 10 or more amino acids. See Fundamental Immunology ch.7 (edited by Paul, 2nd edition, Raven Press, 1989). The variable regions of each light/heavy chain pair form an antibody binding site, in this way a complete immunoglobulin has two binding sites.

Native immunoglobulin chains exhibit the same basic structure in which a relatively conservative framework region (FR) is linked by three highly variable regions, also known as complementarity determining regions or CDRs. From the N terminus to the C terminus, the light and heavy chains contain structural domains FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. The distribution of amino acids in all structural domains is described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th edition, U.S. Dept. Of Health and Human Services, PHS, NIH, NIH Publication No. 91-3242, 1991].

Unless otherwise specified, “antibody” refers to an intact immunoglobulin or antigen-binding portion thereof capable of competing with an intact antibody for specific binding. Antigen-binding moieties can be produced by recombinant DNA techniques, enzymatic or chemical cleavage of intact antibodies. Antigen-binding moieties are, in particular, Fab, Fab', F(ab) ₂ , Fv, structural domain antibodies (dAb), complementarity determining regions (CDR), single chain antibodies (scFv), chimeric antibodies, double chain antibodies (dia Body), a triple chain antibody (triabody), a quadruple chain antibody (tetrabody), and a polypeptide containing at least a portion of an immunoglobulin that binds to a polypeptide-specific antigen.

Fab fragments are monovalent fragments with V _L , V _H , C _L , and C _H1 domains; The F(ab') ₂ fragment is a divalent fragment having two Fab fragments linked by disulfide bonds in the hinge region; Fv fragments have V _H and V _L domains; dAb fragments have an antigen binding fragment of a V _H domain, a V _L domain, or a V _H or V _L domain (US Pat. Nos. US 6,846,634 and US 6,696,245; US Patent Application Publication Nos. US 2005/0202512, US 2004/0202995, US 2004/0038291, US 2004/0009507, and US 2003/0039958; Ward et al. , 1989, Nature 341:544-546).

Single-chain antibody (scFv) is a fusion protein in which the V _L region and the V _H region are joined by a connector (e.g., a synthetic sequence of amino acid residues) to form a continuous protein antibody, wherein the linking group is a protein chain It is long enough to allow it to fold again to form a monovalent antigen-binding site (eg, Bird et al. , 1988, Science 242:423-26) and Huston et al. al. , 1988, Proc. Natl. Acad. Sci. USA 85:5879-83).

A double-chain antibody is a divalent antibody comprising two polypeptide chains, each chain containing V _H and V _L regions linked by a joint that is too short to allow pairing of the two domains in the same chain. Thus, each domain is paired with a complementary domain on another polypeptide chain (eg, Holliger et al. , 1993, Proc. Natl. Acad. Sci. USA 90:6444-48); And Poljak et al. , 1994, Structure 2:11 21-23). If the two polypeptide chains of a double-stranded antibody are identical, the resulting double-stranded antibody due to pairing will have the same antigen-binding site. Polypeptide chains with different sequences can be used to make double-stranded antibodies with different antigen binding sites. Similarly, triple chain and quadruple chain antibodies are antibodies that contain 3 polypeptide chains and 4 polypeptide chains and form 3 antigen binding sites and 4 antigen-binding sites, which may be the same or different.

In the present invention, in order to identify the complementarity determining region (CDR) and framework region (FR) of a given antibody, Kabat et al. [Sequences of Proteins of Immunological Interest, 5th edition, U.S. Dept. Of Health and Human Services, PHS, NIH, NIH Publication No. 91-3242, 1991]. One or more CDRs can be covalently or non-covalently incorporated into a molecule to make it an antibody. Antibodies can incorporate larger polypeptide chains into the CDR(s). The CDR(s) may be covalently attached to another polypeptide chain, or may be non-covalently integrated into the CDR(s). CDRs allow the antibody to specifically bind to a specific antigen of interest.

Antibodies may have one or more binding sites. If more than one binding site is present, the binding site may be the same or different from another binding site. For example, natural human immunoglobulins generally have two identical binding sites, while “bispecific” or “bifunctional” antibodies have two different binding sites.

The term “murine antibody” includes antibodies having one or more variable and constant regions derived from mouse immunoglobulin sequences.

The term "humanized antibody" is an antibody made by grafting the sequence of the complementarity determining region of a mouse antibody molecule into the framework of a human antibody variable region.

The terms "antigen-binding domain", "antigen-binding region", or "antigen-binding site" are portions of antibodies that contain amino acid residues that interact with an antigen and contribute to its specificity and affinity for the antigen. In the case of an antibody that specifically binds an antigen, it will comprise at least a portion of at least one of its CDR domains.

The term “epitope” is the portion of a molecule that binds to (eg, by an antibody) an antibody. Epitopes may contain discontinuous portions of a molecule (e.g., in a polypeptide, amino acid residues that are discontinuous in the primary structure of the polypeptide are bound by antibodies in the tertiary and quaternary structures of the polypeptide, Close enough).

The "percent identity" of two polynucleotide or two polypeptide sequences is determined using the default parameter comparison sequence of the GAP computer program (GCG Wisconsin Package; version 10.3 (part of Accelrys, San Diego, CA)).

The terms "polynucleotide", "oligonucleotide" and "nucleic acid" may alternatively be used throughout the specification, including DNA molecules (eg cDNA or genomic DNA), RNA molecules (eg mRNA), DNA or RNA analogues and nucleotide analogues (eg, peptide nucleic acids and non-natural nucleotide analogues) and their hydrides generated using. Nucleic acid molecules can be single or double stranded. In one embodiment, the nucleic acid molecule encompassed by the present invention encodes an antibody or fragment, derivative, mutant protein, or variant thereof in a continuous open reading frame.

When sequences can be reversed in parallel, the two single-stranded nucleotides are "complementary" to each other, so that each nucleotide in one polynucleotide faces a complementary nucleotide in the other polynucleotide, and the 5'end or 3'of each sequence No gaps are introduced at the ends, and unpaired nucleotides are not found. When two polynucleotides can be interbreed under moderately stringent conditions, one polynucleotide is “complementary” to the other polynucleotide. Thus, one polynucleotide may be complementary to another polynucleotide, but not to its complementary sequence.

The term "carrier" is a nucleic acid that can be used to introduce another nucleic acid linked thereto into a cell. One type of carrier is a “plasmid”, which refers to a linear or circular double-stranded DNA molecule that can be attached to additional nucleic acid segments. Another type of carrier is a viral vector (eg, replication-defective retrovirus, adenovirus, and adenovirus related virus) into which additional DNA segments can be introduced into the viral genome. Some carriers are capable of autonomous replication in the host cell into which it is introduced (eg, bacterial carriers and free-type mammalian carriers containing bacterial origins of replication). Other carriers (e.g., non-free mammalian carriers) integrate into the host cell genome when introduced into the host cell, and thus replicate with the host genome. “Expression carrier” is a type of carrier capable of directing the expression of a selected polynucleotide.

When a regulatory sequence affects the expression (eg, expression level, time, or site) of a nucleotide sequence, the nucleotide sequence is “operably linked” to the regulatory sequence. A “regulatory sequence” is a nucleic acid that affects the expression (eg, level, time, or site of expression) of a nucleic acid operably linked thereto. For example, regulatory genes act directly on the nucleic acid being regulated or through one or more other molecules (eg, regulatory sequences and/or polynucleotides that bind to the nucleic acid). Examples of regulatory sequences include promoters, enhancers, and other expression control elements (eg, polyadenylation signals). Further examples of regulatory sequences are described, for example, in Goeddel, 1990, Gene Expression Technology: Methods in Enzymology , Volume 185, Academic Press, San Diego, CA and Baron et al. , 1995, Nucleic Acids Res . 23:3605-06].

The term “host cell” refers to a cell used to express a nucleic acid as provided herein. Host cells are prokaryotic organisms such as E. Coli, or eukaryotes such as single-celled eukaryotes (e.g. yeast or other fungi), plant cells (e.g. tobacco or tomato plant cells), animal cells (e.g. monkey cells, hamsters) Cells, insect cells, cells of rats and mice) or hybridomas. In general, the host cell is a cultured cell that can be transformed or transfected with a peptide encoding nucleic acid, and such nucleic acid can then be expressed in the host cell. The phrase “recombinant host cell” can be used to describe a host cell transformed or transfected with a nucleic acid to be expressed. A host cell may also be a cell that contains a nucleic acid but does not express the nucleic acid to the desired level unless regulatory sequences have been introduced into the host cell and operably linked to the nucleic acid. The term “host cell” is to be understood to refer to a particular cell of interest as well as progeny or possible progeny of that cell. Due to certain modifications that occur in subsequent generations, such as mutations or environmental influences, the progeny may actually differ from the parent cell, but still fall within the scope of the term used herein.

Long gastric inhibitory peptide receptor

The long gastric inhibitory peptide receptor belongs to type B of the 7-transmembrane G protein-coupled receptor family. This receptor is coupled to one or more intracellular signaling pathways by a heterotrimeric guanine nucleotide binding protein (G protein) (Drucker et al. , 2006, Cell Metab . 3:153-65). To date, studies have shown that GIPR is mainly expressed on the surface of pancreatic β cells and adipocytes (Ravn et al. , 2013, J. Biol. Chem . 288:19760-72), and glucose metabolism and lipid metabolism in humans. It is involved in both, and is therefore closely related to diabetes, obesity and related diseases (Skaw et al. , 2016, Diabetes Obes. Metab . 18:847-854). As used herein, "human GIPR" and "hGIPR" both refer to human enteric inhibitory peptide receptors, which can be used interchangeably. As used herein, "mouse GIPR" and "mGIPR" both refer to an inhibitory peptide receptor on a mouse, and they can also be used interchangeably.

In one embodiment, an antibody provided herein is an antibody that specifically binds to human GIPR. In another embodiment, an antibody provided herein is an antibody that specifically binds to GIPR on a cell membrane, and the antibody is capable of inhibiting or blocking the transmission of GIP signals in such cells. In another embodiment, an antibody provided herein is an antibody that specifically binds to human GIPR and is capable of blocking GIP signaling in these species by binding to GIPR of other species (eg, monkey or mouse). In a further embodiment, an antibody provided herein is a murine antibody that binds to human GIPR and is capable of binding GIPR of another species (eg, monkey).

In one embodiment, the amino acid and polynucleotide sequences of GIPR are from the Gene-Bank database of the US national center for biotechnology information and the Uniprot database of the European institute for biological information. Listed below along with sequence data:

Human ( Homo sapiens ) polynucleotide (SEQ ID NO: 114); Accession number: S79852;

Human (Homo sapiens) amino acid (SEQ ID NO: 113); Accession number: AAB35419.2;

Monkey ( Rhesus macaque ) polynucleotide (SEQ ID NO: 116); Accession number: XM_015124289.1;

Monkey (Rhesus Macaque) Amino Acid (SEQ ID NO: 115); Accession number: XP_014979775;

Mouse ( Mus musculus ) polynucleotide (SEQ ID NO: 118); Accession number: CCDS39795; And mouse (mus musculus) amino acid (SEQ ID NO: 117); Accession number: Q0P543.

Intestinal Gastric Inhibitory Peptide Receptor (GIPR) Antibodies

In one embodiment, GIPR antibodies are provided herein. In another embodiment, the GIPR antibody provided herein is a complete GIPR antibody. In another embodiment, the GIPR antibody provided herein is a GIPR antibody fragment. In another embodiment, a GIPR antibody provided herein is a derivative of a GIPR antibody. In another embodiment, the GIPR antibody provided herein is a GIPR antibody mutant protein. In a further embodiment, a GIPR antibody provided herein is a variant of a GIPR antibody.

In one embodiment, the GIPR antibodies provided herein comprise a sequence of 1, 2, 3, 4, 5, or 6 amino acids, each of which is independently selected from the amino acid sequences listed below:

a. Light chain CDR1 amino acid sequence: SEQ ID NO: 1, SEQ ID NO: 4, SEQ ID NO: 7, SEQ ID NO: 10, SEQ ID NO: 13, and SEQ ID NO: 15;

b. Light chain CDR2 amino acid sequence: SEQ ID NO: 2, SEQ ID NO: 5, SEQ ID NO: 8, SEQ ID NO: 11, and SEQ ID NO: 16;

c. Light chain CDR3 amino acid sequence: SEQ ID NO: 3, SEQ ID NO: 6, SEQ ID NO: 9, SEQ ID NO: 12, SEQ ID NO: 14, and SEQ ID NO: 17;

d. Heavy chain CDR1 amino acid sequence: SEQ ID NO: 18, SEQ ID NO: 23, and SEQ ID NO: 26;

e. Heavy chain CDR2 amino acid sequence: SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 24, SEQ ID NO: 27, and SEQ ID NO: 29; And

f. Heavy chain CDR3 amino acid sequence: SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 25, SEQ ID NO: 28, and SEQ ID NO: 30.

Table 1 lists the amino acid sequences of the light chain CDRs of the GIPR antibodies provided herein, as well as the corresponding polynucleotide coding sequences. Table 2 lists the amino acid sequences of the heavy chain CDRs of the GIPR antibodies provided herein, as well as the corresponding polynucleotide coding sequences.

[Table 1]

Light chain CDR amino acid sequence and polynucleotide coding sequence

[Table 2]

Heavy chain CDR amino acid sequence and polynucleotide coding sequence

In one embodiment, the antibody provided herein is a sequence that differs from one of the CDR amino acid sequences listed in Tables 1 and 2 by addition, replacement, and/or deletion of 5, 4, 3, 2 or 1 single amino acids. Includes. In another embodiment, an antibody provided herein contains a sequence that differs from one of the CDR amino acid sequences listed in Tables 1 and 2 by addition, substitution, and/or deletion of 4, 3, 2 or 1 single amino acids. do.

In another embodiment, an antibody provided herein contains a sequence that differs from one of the CDR amino acid sequences listed in Tables 1 and 2 by 3, 2 or 1 single amino acid addition, replacement, and/or deletion.

In another embodiment, an antibody provided herein contains a sequence that differs from one of the CDR amino acid sequences listed in Tables 1 and 2 by two or one single amino acid addition, replacement, and/or deletion.

In further embodiments, the antibodies provided herein contain a sequence that differs from one of the CDR amino acid sequences listed in Tables 1 and 2 by a single amino acid addition, replacement, and/or deletion.

In one embodiment, a GIPR antibody provided herein comprises a sequence of 1 or 2 amino acids, wherein each amino acid sequence is independently selected from the amino acid sequences listed below:

a. Light chain CDR1 amino acid sequence: SEQ ID NO: 1, SEQ ID NO: 4, SEQ ID NO: 7, SEQ ID NO: 10, SEQ ID NO: 13, and SEQ ID NO: 15; And

b. Heavy chain CDR1 amino acid sequence: SEQ ID NO: 18, SEQ ID NO: 23, and SEQ ID NO: 26.

In another embodiment, a GIPR antibody provided herein comprises a sequence of 1 or 2 amino acids, wherein each amino acid sequence is independently selected from the amino acid sequences listed below:

a. Light chain CDR2 amino acid sequence: SEQ ID NO: 2, SEQ ID NO: 5, SEQ ID NO: 8, SEQ ID NO: 11, and SEQ ID NO: 16; And

b. Heavy chain CDR2 amino acid sequence: SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 24, SEQ ID NO: 27, and SEQ ID NO: 29.

In another embodiment, a GIPR antibody provided herein comprises a sequence of 1, 2, 3, or 4 amino acids, wherein each amino acid sequence is independently selected from the amino acid sequences listed below:

a. Light chain CDR3 amino acid sequence: SEQ ID NO: 3, SEQ ID NO: 6, SEQ ID NO: 9, SEQ ID NO: 12, SEQ ID NO: 14, and SEQ ID NO: 17; And

b. Heavy chain CDR3 amino acid sequence: SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 25, SEQ ID NO: 28, and SEQ ID NO: 30.

In another embodiment, a GIPR antibody provided herein comprises a sequence of 1, 2, 3, or 4 amino acids, wherein each amino acid sequence is independently selected from the amino acid sequences listed below:

a. Light chain CDR1 amino acid sequence: SEQ ID NO: 1, SEQ ID NO: 4, SEQ ID NO: 7, SEQ ID NO: 10, SEQ ID NO: 13, and SEQ ID NO: 15;

b. Heavy chain CDR1 amino acid sequence: SEQ ID NO: 18, SEQ ID NO: 23, and SEQ ID NO: 26;

c. Light chain CDR2 amino acid sequence: SEQ ID NO: 2, SEQ ID NO: 5, SEQ ID NO: 8, SEQ ID NO: 11, and SEQ ID NO: 16; And

d. Heavy chain CDR2 amino acid sequence: SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 24, SEQ ID NO: 27, and SEQ ID NO: 29.

In another embodiment, a GIPR antibody provided herein comprises a sequence of 1, 2, 3, or 4 amino acids, wherein each amino acid sequence is independently selected from the amino acid sequences listed below:

a. Light chain CDR1 amino acid sequence: SEQ ID NO: 1, SEQ ID NO: 4, SEQ ID NO: 7, SEQ ID NO: 10, SEQ ID NO: 13, and SEQ ID NO: 15;

b. Heavy chain CDR1 amino acid sequence: SEQ ID NO: 18, SEQ ID NO: 23, and SEQ ID NO: 26;

c. Light chain CDR3 amino acid sequence: SEQ ID NO: 3, SEQ ID NO: 6, SEQ ID NO: 9, SEQ ID NO: 12, SEQ ID NO: 14, and SEQ ID NO: 17; And

d. Heavy chain CDR3 amino acid sequence: SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 25, SEQ ID NO: 28, and SEQ ID NO: 30.

In further embodiments, the GIPR antibodies provided herein comprise a sequence of 1, 2, 3, or 4 amino acids, wherein each amino acid sequence is independently selected from the amino acid sequences listed below:

a. Light chain CDR2 amino acid sequence: SEQ ID NO: 2, SEQ ID NO: 5, SEQ ID NO: 8, SEQ ID NO: 11, and SEQ ID NO: 16;

b. Heavy chain CDR2 amino acid sequence: SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 24, SEQ ID NO: 27, and SEQ ID NO: 29;

c. Light chain CDR3 amino acid sequence: SEQ ID NO: 3, SEQ ID NO: 6, SEQ ID NO: 9, SEQ ID NO: 12, SEQ ID NO: 14, and SEQ ID NO: 17; And

d. Heavy chain CDR3 amino acid sequence: SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 25, SEQ ID NO: 28, and SEQ ID NO: 30.

In one embodiment, a GIPR antibody provided herein comprises a sequence of 1, 2, or 3 amino acids, wherein each amino acid sequence is independently selected from the amino acid sequences listed below: SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, and SEQ ID NO: 17.

In another embodiment, a GIPR antibody provided herein comprises a sequence of 1, 2, or 3 amino acids, wherein each amino acid sequence is independently selected from the amino acid sequences listed below: SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO : 27, SEQ ID NO: 28, SEQ ID NO: 29, and SEQ ID NO: 30.

In one embodiment, the GIPR antibody provided herein comprises a combination of light and heavy chain CDR1 amino acid sequences independently selected from the following list: SEQ ID NO: 1 and SEQ ID NO: 18, SEQ ID NO: 4 and SEQ ID NO: 18, SEQ ID NO: 7 and SEQ ID NO: 23, SEQ ID NO: 10 and SEQ ID NO: 26, SEQ ID NO: 13 and SEQ ID NO: 26, and SEQ ID NO: 15 and SEQ ID NO : 26.

In another embodiment, the GIPR antibody provided herein comprises a combination of light and heavy chain CDR2 amino acid sequences independently selected from the following list: SEQ ID NO: 2 and SEQ ID NO: 19, SEQ ID NO: 5 and SEQ ID NO: 21, SEQ ID NO: 8 and SEQ ID NO: 24, SEQ ID NO: 11 and SEQ ID NO: 27, and SEQ ID NO: 16 and SEQ ID NO: 29.

In further embodiments, the GIPR antibodies provided herein comprise a combination of light and heavy chain CDR3 amino acid sequences independently selected from the following list: SEQ ID NO: 3 and SEQ ID NO: 20, SEQ ID NO: 6 and SEQ ID NO: 22, SEQ ID NO: 9 and SEQ ID NO: 25, SEQ ID NO: 12 and SEQ ID NO: 28, SEQ ID NO: 14 and SEQ ID NO: 28, and SEQ ID NO: 17 and SEQ ID NO : 30.

In one embodiment, a GIPR antibody provided herein comprises:

a. Combinations of light and heavy chain CDR1 amino acid sequences independently selected from the following list: SEQ ID NO: 1 and SEQ ID NO: 18, SEQ ID NO: 4 and SEQ ID NO: 18, SEQ ID NO: 7 and SEQ ID NO: 23, SEQ ID NO: 10 and SEQ ID NO: 26, SEQ ID NO: 13 and SEQ ID NO: 26, and SEQ ID NO: 15 and SEQ ID NO: 26; And

b. Combinations of light and heavy chain CDR2 amino acid sequences independently selected from the following list: SEQ ID NO: 2 and SEQ ID NO: 19, SEQ ID NO: 5 and SEQ ID NO: 21, SEQ ID NO: 8 and SEQ ID NO: 24, SEQ ID NO: 11 and SEQ ID NO: 27, and SEQ ID NO: 16 and SEQ ID NO: 29.

In another embodiment, a GIPR antibody provided herein comprises:

a. Combinations of light and heavy chain CDR1 amino acid sequences independently selected from the following list: SEQ ID NO: 1 and SEQ ID NO: 18, SEQ ID NO: 4 and SEQ ID NO: 18, SEQ ID NO: 7 and SEQ ID NO: 23, SEQ ID NO: 10 and SEQ ID NO: 26, SEQ ID NO: 13 and SEQ ID NO: 26, and SEQ ID NO: 15 and SEQ ID NO: 26; And

b. Combinations of light and heavy chain CDR3 amino acid sequences independently selected from the following list: SEQ ID NO: 3 and SEQ ID NO: 20, SEQ ID NO: 6 and SEQ ID NO: 22, SEQ ID NO: 9 and SEQ ID NO: 25, SEQ ID NO: 12 and SEQ ID NO: 28, SEQ ID NO: 14 and SEQ ID NO: 28, and SEQ ID NO: 17 and SEQ ID NO: 30.

In another embodiment, a GIPR antibody provided herein contains:

a. Combinations of light and heavy chain CDR2 amino acid sequences independently selected from the following list: SEQ ID NO: 2 and SEQ ID NO: 19, SEQ ID NO: 5 and SEQ ID NO: 21, SEQ ID NO: 8 and SEQ ID NO: 24, SEQ ID NO: 11 and SEQ ID NO: 27, and SEQ ID NO: 16 and SEQ ID NO: 29; And

b. Combinations of light and heavy chain CDR3 amino acid sequences independently selected from the following list: SEQ ID NO: 3 and SEQ ID NO: 20, SEQ ID NO: 6 and SEQ ID NO: 22, SEQ ID NO: 9 and SEQ ID NO: 25, SEQ ID NO: 12 and SEQ ID NO: 28, SEQ ID NO: 14 and SEQ ID NO: 28, and SEQ ID NO: 17 and SEQ ID NO: 30.

In a further embodiment, a GIPR antibody provided herein comprises:

a. Combinations of light and heavy chain CDR1 amino acid sequences independently selected from the following list: SEQ ID NO: 1 and SEQ ID NO: 18, SEQ ID NO: 4 and SEQ ID NO: 18, SEQ ID NO: 7 and SEQ ID NO: 23, SEQ ID NO: 10 and SEQ ID NO: 26, SEQ ID NO: 13 and SEQ ID NO: 26, and SEQ ID NO: 15 and SEQ ID NO: 26;

b. Combinations of light and heavy chain CDR2 amino acid sequences independently selected from the following list: SEQ ID NO: 2 and SEQ ID NO: 19, SEQ ID NO: 5 and SEQ ID NO: 21, SEQ ID NO: 8 and SEQ ID NO: 24, SEQ ID NO: 11 and SEQ ID NO: 27, and SEQ ID NO: 16 and SEQ ID NO: 29; And

c. Combinations of light and heavy chain CDR3 amino acid sequences independently selected from the following list: SEQ ID NO: 3 and SEQ ID NO: 20, SEQ ID NO: 6 and SEQ ID NO: 22, SEQ ID NO: 9 and SEQ ID NO: 25, SEQ ID NO: 12 and SEQ ID NO: 28, SEQ ID NO: 14 and SEQ ID NO: 28, and SEQ ID NO: 17 and SEQ ID NO: 30.

In one embodiment, a GIPR antibody provided herein comprises:

a. Combination of light and heavy chain CDR1, CDR2 and CDR3 amino acid sequences: SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 18, SEQ ID NO: 19, and SEQ ID NO: 20;

b. Combination of light and heavy chain CDR1, CDR2 and CDR3 amino acid sequences: SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 18, SEQ ID NO: 21, and SEQ ID NO: 22;

c. Combination of light and heavy chain CDR1, CDR2 and CDR3 amino acid sequences: SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 23, SEQ ID NO: 24, and SEQ ID NO: 25;

d. Combination of light and heavy chain CDR1, CDR2 and CDR3 amino acid sequences: SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 26, SEQ ID NO: 27, and SEQ ID NO: 28;

e. Combination of light and heavy chain CDR1, CDR2 and CDR3 amino acid sequences: SEQ ID NO: 13, SEQ ID NO: 11, SEQ ID NO: 14, SEQ ID NO: 26, SEQ ID NO: 27, and SEQ ID NO: 28; or

f. Combination of light and heavy chain CDR1, CDR2 and CDR3 amino acid sequences: SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 26, SEQ ID NO: 29, and SEQ ID NO: 30.

In one embodiment, a GIPR antibody provided herein comprises a sequence of 1 or 2 amino acids, wherein each amino acid sequence is independently selected from the amino acid sequences listed below:

a. Light chain variable domain amino acid sequence: SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID NO: 70, and SEQ ID NO: 71; And an amino acid sequence that is at least 80%, at least 85%, at least 90%, or at least 95% identical to any of the above sequences; And

b. Heavy chain variable domain amino acid sequence: SEQ ID NO: 72, SEQ ID NO: 73, SEQ ID NO: 74, SEQ ID NO: 75, SEQ ID NO: 76, SEQ ID NO: 77, SEQ ID NO: 78, SEQ ID NO: 79, and SEQ ID NO: 80; And an amino acid sequence that is at least 80%, at least 85%, at least 90%, or at least 95% identical to any of the above sequences.

In another embodiment, the polynucleotide coding sequence for a GIPR antibody provided herein comprises one or two polynucleotide coding sequences, wherein each polynucleotide coding sequence is independently selected from the polynucleotide sequences listed below:

a. Light chain variable domain polynucleotide coding sequence: SEQ ID NO: 81, SEQ ID NO: 82, SEQ ID NO: 83, SEQ ID NO: 84, SEQ ID NO: 85, SEQ ID NO: 86, SEQ ID NO: 87, SEQ ID NO: 88, SEQ ID NO: 89, SEQ ID NO: 90, and SEQ ID NO: 91; And a polynucleotide sequence that is at least 80%, at least 85%, at least 90%, or at least 95% identical to any of the above sequences; And

b. Heavy chain variable domain polynucleotide coding sequence: SEQ ID NO: 92, SEQ ID NO: 93, SEQ ID NO: 94, SEQ ID NO: 95, SEQ ID NO: 96, SEQ ID NO: 97, SEQ ID NO: 98, SEQ ID NO: 99, and SEQ ID NO: 100; And a polynucleotide sequence that is at least 80%, at least 85%, at least 90%, or at least 95% identical to any of the above sequences.

In one embodiment, the GIPR antibody provided herein comprises an amino acid sequence independently selected from the following list: SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID NO: 70, and SEQ ID NO: 71.

In another embodiment, the GIPR antibodies provided herein comprise an amino acid sequence independently selected from the following list: SEQ ID NO: 72, SEQ ID NO: 73, SEQ ID NO: 74, SEQ ID NO: 75, SEQ ID NO: 76, SEQ ID NO: 77, SEQ ID NO: 78, SEQ ID NO: 79, and SEQ ID NO: 80.

In one embodiment, the GIPR antibody provided herein comprises a combination of amino acid sequences independently selected from the light chain and heavy chain variable domain amino acid sequences listed below: SEQ ID NO: 61 and SEQ ID NO: 72, SEQ ID NO: 62 And SEQ ID NO: 73, SEQ ID NO: 63 and SEQ ID NO: 74, SEQ ID NO: 64 and SEQ ID NO: 74, SEQ ID NO: 65 and SEQ ID NO: 75, SEQ ID NO: 66 and SEQ ID NO: 76, SEQ ID NO: 67 and SEQ ID NO: 77, SEQ ID NO: 68 and SEQ ID NO: 77, SEQ ID NO: 69 and SEQ ID NO: 78, SEQ ID NO: 70 and SEQ ID NO : 79, and SEQ ID NO: 71 and SEQ ID NO: 80.

In one embodiment, the GIPR antibodies provided herein comprise an amino acid sequence independently selected from the following list: SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 73, SEQ ID NO: 74, SEQ ID NO: 76, and SEQ ID NO: 77.

In another embodiment, a GIPR antibody provided herein comprises a combination of amino acid sequences independently selected from the light chain and heavy chain variable domain amino acid sequences listed below: SEQ ID NO: 61 and SEQ ID NO: 72 (L1H1), SEQ ID NO: 62 and SEQ ID NO: 73 (L2H2), SEQ ID NO: 63 and SEQ ID NO: 74 (L3H3), SEQ ID NO: 64 and SEQ ID NO: 74 (L4H3), SEQ ID NO: 65 and SEQ ID NO: 75 (L5H4), SEQ ID NO: 66 and SEQ ID NO: 76 (L6H5), SEQ ID NO: 67 and SEQ ID NO: 77 (L7H6), SEQ ID NO: 68 and SEQ ID NO: 77 (L8H6), SEQ ID NO: 69 and SEQ ID NO: 78 (L9H7), SEQ ID NO: 70 and SEQ ID NO: 79 (L10H8), and SEQ ID NO: 71 and SEQ ID NO: 80 (L11H9).

The symbol “LxHy” may also be used herein to refer to a GIPR antibody provided herein, where “x” corresponds to a light chain variable region sequence code and “y” corresponds to a heavy chain variable region sequence code. For example, L2H2 is a complete antibody having a light chain variable region comprising the amino acid sequence of SEQ ID NO: 62(L2) and a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 73(H2).

In one embodiment, a GIPR antibody provided herein comprises a sequence of 1 or 2 amino acids, wherein each amino acid sequence is independently selected from the amino acid sequences listed below:

a. Light chain constant amino acid sequence: SEQ ID NO: 101 and SEQ ID NO: 102; And

b. Heavy chain constant amino acid sequence: SEQ ID NO: 103 and SEQ ID NO: 104, and SEQ ID NO: 124.

In one embodiment, the GIPR antibodies provided herein comprise a sequence of 1 or 2 amino acids, wherein each amino acid sequence is independently selected from a combination of light and heavy chain constant amino acid sequences listed below: SEQ ID NO: 101 and SEQ ID NO: 103, SEQ ID NO: 101 and SEQ ID NO: 104, SEQ ID NO: 102 and SEQ ID NO: 103, and SEQ ID NO: 102 and SEQ ID NO: 104. In another embodiment, the present application The GIPR antibody provided in comprises one or two amino acid sequences, wherein each amino acid sequence is independently selected from a combination of light and heavy chain constant amino acid sequences listed below: SEQ ID NO: 101 and SEQ ID NO: 124, And SEQ ID NO: 102 and SEQ ID NO: 124.

In one embodiment, the GIPR antibodies provided herein comprise the amino acid sequences of the light and heavy chain CDRs and FRs (framework) listed herein. The amino acid sequence of the FR is contained in the light or heavy chain variable domain and is not separately indicated. In one embodiment, the antibody comprises a light chain CDR1 sequence listed herein. In another embodiment, the antibody comprises a light chain CDR2 sequence listed herein. In another embodiment, the antibody comprises a light chain CDR3 sequence listed herein. In another embodiment, the antibody comprises a heavy chain CDR1 sequence listed herein. In another embodiment, the antibody comprises a heavy chain CDR2 sequence listed herein. In another embodiment, the antibody comprises a heavy chain CDR3 sequence listed herein. In another embodiment, the antibody comprises a light chain FR1 sequence herein. In another embodiment, the antibody comprises a light chain FR2 sequence herein. In another embodiment, the antibody comprises a light chain FR3 sequence herein. In another embodiment, the antibody comprises a light chain FR4 sequence herein. In another embodiment, the antibody comprises a heavy chain FR1 sequence herein. In another embodiment, the antibody comprises a heavy chain FR2 sequence herein. In another embodiment, the antibody comprises a heavy chain FR3 sequence herein. In a further embodiment, the antibody comprises a heavy chain FR4 sequence herein.

In one embodiment, the light chain CDR3 sequence of the antibody is described above by addition(s), substitution(s) and/or deletion(s) of 6, 5, 4, 3, 2 or 1 or less amino acids. It differs from SEQ ID NO: 6, SEQ ID NO: 12, and SEQ ID NO: 14 of the illustrated light chain CDR3 sequence. In another embodiment, the heavy chain CDR3 sequence of the antibody is by addition(s), substitution(s) and/or deletion(s) of 6, 5, 4, 3, 2 or 1 or less amino acids. It is different from SEQ ID NO: 22 and SEQ ID NO: 28 of the heavy chain CDR3 sequence exemplified above. In a further embodiment, the light chain CDR3 sequence of the antibody is said by addition(s), substitution(s) and/or deletion(s) of 6, 5, 4, 3, 2 or 1 or less amino acids. Different from SEQ ID NO: 6, SEQ ID NO: 12, and SEQ ID NO: 14 of the illustrated light chain CDR3 sequence, and the heavy chain CDR3 sequence of the antibody is 6, 5, 4, 3, 2 or 1 It differs from SEQ ID NO: 22 and SEQ ID NO: 28 of the heavy chain CDR3 sequence illustrated above by no more than 4 amino acid addition(s), substitution(s) and/or deletion(s). In another embodiment, the antibody further comprises 1, 2, 3, 4, 5 or 6 combinations of the light and heavy chain CDR sequences illustrated above.

In one embodiment, the GIPR antibodies provided herein are L2 (SEQ ID NO: 62), L3 (SEQ ID NO: 63), L4 (SEQ ID NO: 64), L6 (SEQ ID NO: 66), as listed herein, A light chain variable domain amino acid sequence selected from L7 (SEQ ID NO: 67), and L8 (SEQ ID NO: 68) light chain variable domain sequences. In one embodiment, the amino acid sequence of the light chain variable domain of the GIPR antibody is L2 (SEQ ID NO: 62), L3 (SEQ ID NO: 63), L4 (SEQ ID NO: 64), L6 (SEQ ID NO: 66) , L7 (SEQ ID NO: 67), and L8 (SEQ ID NO: 68) of one light chain variable domain amino acid sequence and 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 amino acids are different, wherein each sequence difference is independently a deletion, insertion or substitution of an amino acid residue. In another embodiment, the light chain variable domain of the GIPR antibody is L2 (SEQ ID NO: 62), L3 (SEQ ID NO: 63), L4 (SEQ ID NO: 64), L6 (SEQ ID NO: 66), L7 (SEQ ID NO: 67), and at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% of the amino acid sequence of the light chain variable domain of one of L8 (SEQ ID NO: 68) , At least 97%, or at least 99% identical amino acid sequences. In another embodiment, the polynucleotide coding sequence of the light chain variable domain of the GIPR antibody is L2 (SEQ ID NO: 62), L3 (SEQ ID NO: 63), L4 (SEQ ID NO: 64), L6 (SEQ ID NO. : 66), at least 70%, at least 75%, at least 80%, at least 85%, at least 90% with the polynucleotide coding sequence of one of L7 (SEQ ID NO: 67), and L8 (SEQ ID NO: 68), At least 95%, at least 97%, or at least 99% identical nucleotide coding sequences. In another embodiment, the polynucleotide coding sequence of the light chain variable domain of the GIPR antibody is L2 (SEQ ID NO: 62), L3 (SEQ ID NO: 63), L4 (SEQ ID NO: 64), L6 (SEQ ID NO. : 66), the complementary polynucleotide coding sequence of one of L7 (SEQ ID NO: 67), and L8 (SEQ ID NO: 68) and a polynucleotide sequence that hybridizes under appropriate conditions. In a further embodiment, the polynucleotide coding sequence of the light chain variable domain of the GIPR antibody is L2 (SEQ ID NO: 62), L3 (SEQ ID NO: 63), L4 (SEQ ID NO: 64), L6 (SEQ ID NO: 66), the complementary polynucleotide coding sequence of the light chain variable domain of one of L7 (SEQ ID NO: 67), and L8 (SEQ ID NO: 68) and a polynucleotide sequence that hybridizes under stringent conditions.

In one embodiment, the GIPR antibodies provided herein are H2 (SEQ ID NO: 73), H3 (SEQ ID NO: 74), H5 (SEQ ID NO: 76), and H6 (SEQ ID NO: 77) listed herein. It includes a heavy chain variable domain amino acid sequence selected from heavy chain variable domain sequences. In another embodiment, the heavy chain variable domain amino acid sequence of the GIPR antibody is H2 (SEQ ID NO: 73), H3 (SEQ ID NO: 74), H5 (SEQ ID NO: 76), and H6 (SEQ ID NO: 77). ) Of one heavy chain variable domain sequence and 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 Or one amino acid is different, wherein the difference in each sequence is independently a deletion, insertion or substitution of one amino acid residue. In another embodiment, the heavy chain variable domain of the GIPR antibody is among H2 (SEQ ID NO: 73), H3 (SEQ ID NO: 74), H5 (SEQ ID NO: 76), and H6 (SEQ ID NO: 77). An amino acid sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identical to one heavy chain variable domain sequence. In another embodiment, the heavy chain variable domain of the GIPR antibody is among H2 (SEQ ID NO: 73), H3 (SEQ ID NO: 74), H5 (SEQ ID NO: 76), and H6 (SEQ ID NO: 77). Comprises a polynucleotide coding sequence that is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% identical to one heavy chain variable domain polynucleotide coding sequence . In another embodiment, the polynucleotide coding sequence of the GIPR antibody heavy chain variable domain is H2 (SEQ ID NO: 73), H3 (SEQ ID NO: 74), H5 (SEQ ID NO: 76), and H6 under moderately stringent conditions. (SEQ ID NO: 77) a polynucleotide sequence that hybridizes to the complementary polynucleotide coding sequence of one heavy chain variable domain. In one embodiment, the polynucleotide coding sequence of the GIPR antibody heavy chain variable domain is H2 (SEQ ID NO: 73), H3 (SEQ ID NO: 74), H5 (SEQ ID NO: 76), and H6 (SEQ ID NO: 77) a complementary polynucleotide coding sequence of one of the heavy chain variable domains and a polynucleotide sequence that hybridizes under stringent conditions.

In one embodiment, the antibodies provided herein are L1H1 (SEQ ID NO: 61 and SEQ ID NO: 72), L2H2 (SEQ ID NO: 62 and SEQ ID NO: 73), L3H3 (SEQ ID NO: 63 and SEQ ID NO: 73). NO: 74), L4H3 (SEQ ID NO: 64 and SEQ ID NO: 74), L5H4 (SEQ ID NO: 65 and SEQ ID NO: 75), L6H5 (SEQ ID NO: 66 and SEQ ID NO: 76), L7H6 (SEQ ID NO: 67 and SEQ ID NO: 77), L8H6 (SEQ ID NO: 68 and SEQ ID NO: 77), L9H7 (SEQ ID NO: 69 and SEQ ID NO: 78), L10H8 (SEQ ID NO: : 70 and SEQ ID NO: 79), or a combination of L11H9 (SEQ ID NO: 71 and SEQ ID NO: 80), or a desired phenotype (e.g., IgA, IgG1, IgG2a, IgG2b, IgG3, IgM, IgE, Or a combination of IgD), or a Fab or F(ab')2 fragment thereof.

In one embodiment, the antibodies provided herein are L2H2 (SEQ ID NO: 62 and SEQ ID NO: 73), L3H3 (SEQ ID NO: 63 and SEQ ID NO: 74), L4H3 (SEQ ID NO: 64 and SEQ ID NO: NO: 74), L6H5 (SEQ ID NO: 66 and SEQ ID NO: 76), L7H6 (SEQ ID NO: 67 and SEQ ID NO: 77), or L8H6 (SEQ ID NO: 68 and SEQ ID NO: 77) Or a combination of the desired phenotype (eg, IgA, IgG1, IgG2a, IgG2b, IgG3, IgM, IgE or IgD), or a Fab or F(ab')2 fragment thereof.

Antibodies provided herein may comprise any constant region known in the art. The light chain constant region can be, for example, a κ or λ light chain constant region, such as a mouse κ or λ light chain constant region. The heavy chain constant region may be, for example, an α, δ, ε, γ, or μ heavy chain constant region, such as a mouse α, δ, ε, γ, or μ heavy chain constant region. In one embodiment, the light or heavy chain constant region is a fragment, derivative, variant, or mutant of the natural constant region.

In one embodiment, an antibody provided herein further comprises a human light chain κ or λ constant domain or fragment thereof. The amino acid sequence of the light chain constant region is as follows:

Human Light Chain κ Constant Domain Amino Acid Sequence: (SEQ ID NO: 101); And

Human Light Chain λ Constant Domain Amino Acid Sequence: (SEQ ID NO: 102).

In one embodiment, an antibody provided herein further comprises a human heavy chain constant domain or fragment thereof.

The amino acid sequence of the heavy chain constant region is as follows:

Human Heavy Chain Constant Region Amino Acid Sequence (hIgG2): (SEQ ID NO: 103);

Human Heavy Chain Constant Region Amino Acid Sequence (hIgG4): (SEQ ID NO: 104); And

Human Heavy Chain Constant Region Amino Acid Sequence (hIgG4): (SEQ ID NO: 124).

In one embodiment, the GIPR antibodies provided herein are mouse-derived antibodies, humanized antibodies, chimeric antibodies, monoclonal antibodies, polyclonal antibodies, recombinant antibodies, antigen-binding antibody fragments, single chain antibodies, double chain antibodies, triple Chain antibodies, quadruple chain antibodies, Fab fragments, F(ab')x fragments, structural domain antibodies, IgD antibodies, IgE antibodies, IgM antibodies, IgGl antibodies, IgG2 antibodies, IgG3 antibodies, or IgG4 antibodies.

In one embodiment, the GIPR antibody provided herein is a GIPR monoclonal antibody.

In another embodiment, the GIPR antibody provided herein is a monoclonal antibody comprising a combination of amino acid sequences selected from the following list: SEQ ID NO: 61 and SEQ ID NO: 72, SEQ ID NO: 62 and SEQ ID NO: 73, SEQ ID NO: 63 and SEQ ID NO: 74, SEQ ID NO: 64 and SEQ ID NO: 74, SEQ ID NO: 65 and SEQ ID NO: 75, SEQ ID NO: 66 and SEQ ID NO: 76, SEQ ID NO: 67 and SEQ ID NO: 77, SEQ ID NO: 68 and SEQ ID NO: 77, SEQ ID NO: 69 and SEQ ID NO: 78, SEQ ID NO: 70 and SEQ ID NO: 79, And SEQ ID NO: 71 and SEQ ID NO: 80.

In one embodiment, the GIPR antibody provided herein is a mouse GIPR antibody. In another embodiment, the GIPR antibody provided herein is a humanized GIPR antibody.

In one embodiment, the GIPR antibodies provided herein reduce human GIP signaling with an IC50 value of about 1 nM to 200 nM or about 1 nM to 100 nM.

Antibodies and antibody fragments

In one embodiment, an antibody provided herein is a full length antibody (including polyclonal antibodies, monoclonal antibodies, chimeric antibodies, humanized antibodies or human antibodies having full length heavy and/or light chains). In another embodiment, an antibody provided herein is an antibody fragment, e.g., F(ab')2, Fab, Fab', Fv, Fc, or Fd fragment, and is a single domain antibody, single chain antibody, maxibody ( maxibody), minibody, intrabody, double chain antibody, triple chain antibody, quadruple chain antibody, v-NAR and bis-scFv (see, e.g. Hollinger and Hudson, 2005, Nature Biotechnology , 23:1126-1136). In another embodiment, an antibody provided herein also comprises an antibody polypeptide such as disclosed in US Pat. No. 6703199, including a fibronectin polypeptide monobody. In another embodiment, the antibodies provided herein also include other antibody polypeptides disclosed in US Patent Publication 2005/0238646 that are single chain polypeptides.

In one embodiment, the variable region of the IgG gene expressing the monoclonal antibody of interest in the hybridoma is amplified using nucleotide primers. Such primers can be synthesized by one of ordinary skill in the art, or can be purchased from commercially available suppliers, which suppliers in particular for the V _Ha , V _Hb , V _Hc , V _Hd , C _H1 , V _L and C _L regions. Primers for mouse and human variable regions, including primers, are synthesized. These primers can be used to amplify heavy or light chain variable regions, and these regions can then be inserted into vectors such as IMMUNOZAP ^™ H or IMMUNOZAP ^™ L (Stratagene), respectively. Subsequently, these vectors were used for expression in E. Coli, yeast, or mammalian-based systems. Large quantities of single chain proteins containing fusions of the V _H and V _L regions can be produced using this method (see Bird et al. , 1988, Science 242:423-426).

One of skill in the art will understand that certain proteins, such as antibodies, may undergo various post-translational modifications. The type and extent of these modifications often depend on the culture conditions as well as the host cell line used to express the protein. Such modifications may include changes in glycosylation, methionine oxidation, diketopiperazine formation, aspartate isomerization and asparagine deamidation. A frequent modification is the loss of carboxyl-terminal basic residues (such as lysine or arginine) due to the action of carboxypeptidases (as described in Harris, 1995, Journal of Chromatography 705:129-134).

A common method for the production of murine monoclonal antibodies is by hybridoma cells. Monoclonal antibodies can be isolated and purified by a variety of well-established techniques. Such isolation techniques include affinity chromatography with Protein-A Sepharose, size-exclusion chromatography, and ion-exchange chromatography (see, for example, Coligan at pages 2.7.1-2.7.12 and pages). 2.9.1-2.9.3]; Baines et al. , "Purification of Immunoglobulin G (IgG)," in Methods in Molecular Biology, Vol. 10, pages 79-104 (The Humana Press, Inc. 1992)] Reference). Monoclonal antibodies can be purified by affinity chromatography using an appropriate ligand selected based on the specific properties of the antibody (eg, heavy or light chain isotype, binding specificity, etc.). Examples of suitable ligands immobilized on a solid support include protein A, protein G, anti-constant region (light or heavy chain) antibodies, anti-idiotype antibodies, and TGF-β binding proteins, or fragments or variants thereof.

In addition, the molecular evolution of the complementarity determining region (CDR) at the center of the antibody binding site has been attributed to antibodies with increased affinity, such as Schier et al. , 1996, J. Mol. Biol. 263:551-567] was used to isolate antibodies with increased affinity for c-erbB-2. Thus, such a technique is useful for making antibodies against GIPR.

Antibodies to human GIPR can be used, for example, in assays to detect the presence of GIPR in vitro or in vivo.

Antibodies can also be made by any conventional technique. For example, such antibodies can be purified from cells that naturally express them (e.g., antibodies can be purified from hybridomas that produce them), or in recombinant expression systems using any technique known in the art. Can be produced. See, eg, Monoclonal Antibodies, Hybridomas: A New Dimension in Biological Analyses, Kennet et al. (eds.), Plenum Press, New York (1980)]; And Antibodies: A Laboratory Manual, Harlow and Land (eds.), Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, (1988). This is discussed in the nucleic acid section below.

Antibodies can be prepared and screened for desired properties by any known technique. Some techniques involve the isolation of nucleic acids encoding the polypeptide chains (or portions thereof) of related antibodies (eg, anti-GIPR antibodies), and manipulation of nucleic acids. A nucleic acid can be fused with another related nucleic acid or modified by recombinant DNA techniques (eg, mutagenesis or other conventional techniques) to add, delete, or replace one or more amino acid residues.

When it is desired to improve the affinity of an antibody according to the invention containing one or more of the above-mentioned CDRs, such antibodies may be used to maintain the CDRs (Yang et al. , 1995, J. Mol. Biol. , 254) . :392-403]), chain shuffling (Marks et al. , 1992, Bio/Technology , 10:779-783), E. Use of mutant strains of E. coli (Low et al. , 1996, J. Mol. Biol. , 250:350-368), DNA shuffling (Patten et al. , 1997, Curr. Opin. Biotechnol. , 8:724-733]), phage display (Thompson et al. , 1996, J. Mol. Biol. , 256:7-88) and additional PCR techniques (Crameri et al., 1998, Nature , 391:288-291]) can be obtained by a number of affinity maturation protocols. Both of these methods or affinity maturation are discussed in Vaughan et al ., 1998, Nature Biotechnology , 16:535-539.

In one embodiment, a fragment of a GIPR antibody is provided herein. Such fragments may comprise entirely antibody-derived sequences or additional sequences. Examples of antigen binding fragments include Fab, F(ab')2, single chain antibodies, diabodies, tribodies, tetrabodies, and domain antibodies. Another example is described in Lunde et al. , 2002, Biochem. Soc. Trans. 30:500-06].

Single chain antibodies can be formed by linking heavy and light chain variable domain (Fv regions) fragments through amino acid bridges (short peptide linkers) to produce a single polypeptide chain. Such single chain Fv (scFv) was prepared by fusion DNA encoding a peptide linker between DNA encoding two variable domain polypeptides (VL and VH). The resulting polypeptides can themselves be refolded to form antigen-binding monomers, or multimers (e.g., dimers, trimers, or tetramers) depending on the length of the flexible linker between the two variable domains. (Kortt et al. , 1997, Prot. Eng. 10:423); Kortt et al. , 2001, Biomol. Eng. 18:95-108). By combining various VL and VH-comprising polypeptides, multimeric scFvs that bind to various epitopes can be formed (Kriangkum et al. , 2001, Biomol. Eng. 18:31-40). Techniques developed for the production of single chain antibodies include US Pat. Nos. 4946778; Bird, 1988, Science 242:423; Huston et al. , 1988, Proc. Natl. Acad. Sci. USA 85:5879]; Ward et al. , 1989, Nature 334:544]; De Graaf et al. , 2002, Methods Mol. Biol. 178:379-87. Single chain antibodies derived from the antibodies provided herein, including, but not limited to, scFvs comprising the variable domain combination, L1H1, are included in the present invention.

Antibodies derived from antibodies can also be obtained, for example, by proteolytic hydrolysis of the antibody, for example pepsin or papain digestion of whole antibodies according to conventional methods. As an example, antibody fragments can be produced by enzymatic cleavage of antibodies with pepsin to give SS fragments designated F(ab')2. This fragment can be further cleaved using a thiol reducing agent to produce a 3.5S Fab' monovalent fragment. Optionally, the cleavage reaction can be carried out using a blocker for the sulfhydryl group resulting from cleavage of the disulfide bond. As an alternative, enzymatic cleavage with papain directly produces two monovalent Fab fragments and an Fc fragment. This method is described, for example, in US Pat. No. 4331647 to Goldenberg, Nisonoffet et al. , 1960, Arch. Biochem. Biophys. 89:230]; Porter, 1959, Biochem. J. 73:119]; Edelman et al. , Methods in Enzymology 1:422 (Academic Press 1967)]; And Andrews, SM and Titus, JA in Current Protocols in Immunology (Coligan JE, et al. , eds), John Wiley & Sons, New York (2003), pages 2.8.1-2.8.10 and 2.10 A.1-2.10A.5]. As long as the fragment binds to the antigen recognized by the intact antibody, other antibody cleavage methods, such as separating the heavy chain to form a monovalent light-heavy chain fragment (Fd), further cleavage of the fragment, or other enzymatic, chemical , Or genetic techniques can also be used.

Another form of antibody fragment is a peptide comprising one or more complementarity determining regions (CDRs) of an antibody. CDRs can be obtained by constructing polynucleotides encoding CDRs. Such polynucleotides are prepared, for example, by using a polymerase chain reaction to synthesize variable regions using mRNA or antibody-producing cells as templates (see, e.g. Larrick et al. , 1991, Methods). : A Companion to Methods in Enzymology 2:106]; Courtenay-Luck, "Genetic Manipulation of Monoclonal Antibodies," in Monoclonal Antibodies: Production, Engineering and Clinical Application, Ritter et al. (eds.), page 166 (Cambridge) University Press 1995); and Ward et al. , "Genetic Manipulation and Expression or Antibodies," in Monoclonal Antibodies: Principles and Applications, Birch et al. , (eds.), page 137 (Wiley-Liss, Inc. 1995)]. The antibody fragment may further comprise at least one variable region domain of an antibody described herein. Thus, for example, the V region domain is a monomer and may be a V _H or V _L domain, which can bind to GIPR with an affinity of 1 x 10 ^-7 M or less, as described below.

The variable region domain can be any naturally occurring variable domain or an engineered version thereof. Engineered version refers to the variable region domain generated using recombinant DNA engineering techniques. Such engineered versions include those generated from a specific antibody variable region, for example by insertion, deletion, or alteration in or to the amino acid sequence of a specific antibody. Specific examples include engineered variable region domains containing at least one CDR from a first antibody and optionally one or more framework amino acids and the remainder of the variable region domain from a second antibody.

The variable region domain may be covalently attached to at least one other antibody domain or fragment thereof at the C-terminal amino acid. Thus, for example, the V _H domain present in the variable region domain can be linked to an immunoglobulin C _H1 domain or fragment thereof. Similarly, the V _L domain can be linked to the C _k domain or a fragment thereof. In this way, for example, the antibody may be a Fab fragment, wherein the antigen binding domain is C _H1 and Cκ at the C-terminus It contains related V _H and V _L domains that are each covalently linked to the domain. The C _H1 domain can be extended with additional amino acids to provide, for example, a hinge region or a portion of a hinge region domain as found in Fab' fragments, or to provide additional domains such as antibody C _H2 and C _H3 domains. .

Derivatives and variants of antibodies

The nucleotide sequences of L1 and H1 can be random mutagenesis or site-directed, e.g., to generate altered polynucleotides comprising one or more specific nucleotide substitutions, deletions, or insertions compared to unmutated polynucleotides. directed) mutagenesis (eg, oligonucleotide-directed site-specific mutagenesis). Examples of techniques for making such alterations are described in Walder et al. , 1986, Gene 42:133]; Bauer et al. , 1985, Gene 37:73]; Kraik, 1985, BioTechniques , 3:12-19; See Smith et al. , 1981, Genetic Engineering: Principles and Methods, Plenum Press]; And in U.S. Patent Nos. 4518584 and 477462. For example, these and other methods can be used to increase the affinity, avidity, or specificity for GIPR compared to non-derivatized antibodies, or to increase stability in vivo or in vitro, or reduce side effects in vivo. It can be used to create derivatives of GIPR antibodies with properties.

Other derivatives of GIPR antibodies within the scope of the invention include, for example, anti-GIPR antibodies, by expression of a recombinant fusion protein comprising a heterologous polypeptide fused to the N-terminus or C-terminus of an anti-GIPR antibody polypeptide, or Fragments thereof and covalent or aggregated conjugates of other proteins or polypeptides are included. For example, the conjugated peptide may be a heterologous signal (or leader) polypeptide, such as a yeast alpha-factor leader or peptide, such as an epitope tag. The antibody containing the fusion protein may include a peptide added to facilitate purification or identification of an antigen binding protein (eg, poly-His). Antibodies are also described in Hopp et al. , 1988, Bio/Technology 6:1204] and U.S. Patent No. 5011912. FLAG peptides are highly antigenic and provide epitopes that are reversibly bound by specific monoclonal antibodies (mAbs), allowing rapid assays and easy purification of expressed recombinant proteins. Reagents useful for preparing fusion proteins in which the FLAG peptide is fused to a given polypeptide are commercially available (Sigma, St. Louis, Mo.). In another embodiment, oligomers containing one or more antibodies can be used as GIPR antagonists. Oligomers can be in the form of covalently linked or non-covalently linked dimers, trimers, or higher oligomers. Oligomers comprising two or more antibodies are contemplated for use, one example being a homodimer. Other oligomers include heterodimer, homotrimer, heterotrimer, homotetramer, and heterotetramer.

One embodiment relates to an oligomer comprising a plurality of antibodies bound through covalent or non-covalent interactions between peptide moieties fused to the antibody. Such a peptide may be a peptide linker (spacer), or a peptide having properties that promote oligomerization. As described in more detail below, among the peptides capable of promoting oligomerization of antibodies attached to the peptides are leucine zippers, and certain polypeptides derived from antibodies.

In certain embodiments, the oligomer comprises 2-4 antibodies. The oligomeric antibody may be of any form described above, for example, any form such as a variant or fragment. Preferably, the oligomer comprises an antibody that exhibits GIPR binding activity.

In one embodiment, the oligomer is prepared using a polypeptide derived from an immunoglobulin. The preparation of fusion proteins comprising specific heterologous polypeptides fused to various portions (including Fc domains) of antibody-derived polypeptides is described, for example, in Ashkenazi et al ., 1991, PNAS USA 88:10535; Byrn et al. , 1990, Nature 344:677]; And Holenbaugh et al. , 1992 "Construction of Immunoglobulin Fusion Proteins", in Current Protocols in Immunology, Suppl. 4, pages 10.19.1-10.19.11]. One embodiment provided herein relates to a dimer comprising two fusion proteins produced by fusing a GIPR binding fragment of an anti-GIPR antibody to the Fc region of an antibody. Dimers, for example, insert a gene fusion encoding the fusion protein into an appropriate expression vector, express the gene fusion in a host cell transformed with the recombinant expression vector, and make the expressed fusion protein very similar to an antibody molecule. It can be made by allowing it to assemble, resulting in the formation of interchain disulfide bonds between the Fc moieties, resulting in a dimer.

The term “Fc polypeptide” as used herein includes natural and mutein forms of polypeptides derived from the Fc region of an antibody. Also included are truncated forms of such polypeptides containing hinge regions that promote dimerization. Fusion proteins comprising Fc moieties (and oligomers formed therefrom) provide the advantage of being readily purified by affinity chromatography on a Protein A or Protein G column.

One suitable Fc polypeptide described in PCT application WO 93/10151 (which is incorporated herein by reference) is a single chain polypeptide extending from the N-terminal hinge region of a human IgG1 antibody to the native C-terminus of the Fc region. Another useful Fc polypeptide is described in US Pat. No. 5457035 and Baum et al. , 1994, EMBO J. 13:3992-4001]. The amino acid sequence of this mutein is presented in WO 93/10151, except that amino acid 19 is changed from Leu to Ala, amino acid 20 is changed from Leu to Glu, and amino acid 22 is changed from Gly to Ala. It is identical to the amino acid sequence of the native Fc sequence. Mutains show a reduced affinity for the Fc receptor. In other embodiments, the variable portion of the heavy and/or light chain of the anti-GIPR antibody may replace the variable portion of the heavy and/or light chain of the antibody.

Alternatively, oligomers are fusion proteins comprising multiple antibodies with or without peptide linkers (spacer peptides). Among suitable peptide linkers are those described in US Pat. Nos. 4751180 and 4935233.

Another method of making oligomeric antibodies involves the use of leucine zippers. The leucine zipper domain is a peptide that promotes oligomerization of the protein in which it is found. Leucine zippers were first identified in several DNA-binding proteins (Landschulz et al. , 1988, Science 240:1759), and have since been found in several different proteins. Among the known leucine zippers are naturally occurring peptides and derivatives thereof that are dimerized or trimerized. Examples of leucine zipper domains suitable for producing soluble oligomeric proteins are described in PCT application WO 94/10308, and leucine zippers derived from lung surfactant protein D (SPD) are incorporated herein by reference [Hoppe et al. . , 1994, FEBS Letters 344:191. The use of a modified leucine zipper that allows stable trimerization of the heterologous protein fused thereto is described in Fanslow et al. , 1994, Semin. Immunol. 6:267-78. In one method, a recombinant fusion protein comprising an anti-GIPR antibody fragment or derivative fused to a leucine zipper peptide is expressed in a suitable host cell, and the resulting soluble oligomeric anti-GIPR antibody fragment or derivative is recovered from the culture supernatant.

In yet another embodiment, the antibody derivative may comprise at least one of the CDRs disclosed herein. For example, one or more CDRs can be incorporated into known antibody framework regions (IgG1, IgG2, etc.) or conjugated to a suitable vehicle to enhance its half-life. Suitable vehicles include, but are not limited to, Fc, albumin, transferrin, and the like. Such vehicles and other suitable vehicles are known in the art. Such conjugated CDR peptides may be monomeric, dimer, tetramer, or other forms. In one embodiment, one or more water-soluble polymers are bonded at one or more specific positions of the binder, for example at the amino terminus. In one example, the antibody derivative comprises one or more water soluble polymer attachments including, but not limited to, polyethylene glycol, polyoxyethylene glycol, or polypropylene glycol. See, for example, U.S. Patent Nos. 4640835, 4496689, 4301144, 4670417, 4791192 and 417337. In certain embodiments, the derivative is monomethoxy-polyethylene glycol, dextran, cellulose, or other carbohydrate based polymer, poly-( N -vinyl pyrrolidone)-polyethylene glycol, propylene glycol homopolymer, polypropylene oxide/ethylene oxide Copolymers, polyoxyethylated polyols (eg glycerol) and polyvinyl alcohol, as well as mixtures of such polymers. In certain embodiments, one or more water soluble polymers are randomly attached to one or more side chains. In certain embodiments, PEG can act to improve the therapeutic capacity of a binding agent such as an antibody. Such specific methods are discussed, for example, in U.S. Patent No. 6133426, which is incorporated herein by reference for any purpose.

It will be appreciated that the antibodies provided herein may have at least one amino acid substitution if the antibody retains binding specificity. Accordingly, modifications to the antibody structure are included within the scope of the present invention. This may include amino acid substitutions that may be conservative or non-conservative that do not impair the human GIPR binding ability of the antibody. Conservative amino acid substitutions may include non-naturally occurring amino acid residues that are typically incorporated by chemical peptide synthesis rather than synthesis in biological systems. It includes peptidomimetic and other reversed or reversed forms of amino acid moieties. Conservative amino acid substitutions may also involve substitution of natural amino acid residues by normative residues, with little or no effect on the polarity or charge of the amino acid residue at that position. Non-conservative substitutions may entail the exchange of members of one class of amino acids or amino acid mimetics for members of another class with different physical properties (e.g., size, polarity, hydrophobicity, charge). have.

Moreover, one of skill in the art can create variants to be tested comprising a single amino acid substitution at each desired amino acid residue. The variants can then be screened using activity assays known to those of skill in the art. Such variants can be used to gather information about suitable variants. For example, if a change to a particular amino acid residue is found to be impaired, undesirably reduced, or result in inappropriate activity, variants with such change can be avoided. That is, based on the information collected from such routine experimentation, one of ordinary skill in the art can easily determine which amino acids should avoid further substitutions made alone or in combination with other mutations.

One of skill in the art will be able to determine suitable variants of the polypeptides as presented herein using well-known techniques. In certain embodiments, one of skill in the art can identify suitable regions of a molecule that can be altered without compromising activity by targeting regions that are not critical for activity. In certain embodiments, residues or portions of a molecule that are conserved between similar polypeptides can be identified. In certain embodiments, conservative amino acid substitutions can occur without compromising biological activity or adversely affecting the structure of the polypeptide, even in regions that may be important for biological activity or structure. Additionally, one of ordinary skill in the art may review structure-function studies to identify residues in similar polypeptides that are important for activity or structure. In view of such a comparison, the importance of an amino acid residue in a protein corresponding to an amino acid residue important for activity or structure in a similar protein can be predicted. One of skill in the art can select chemically similar amino acid substitutions for important amino acid residues so predicted.

One of skill in the art can also analyze the three-dimensional structure and amino acid sequence in relation to its structure in similar polypeptides. Given such information, one of skill in the art can predict the alignment of amino acid residues of an antibody to a three-dimensional structure. In certain embodiments, one of skill in the art may choose not to make radical alterations to amino acid residues that are predicted to be on the surface of the protein, as such residues may be involved in important interactions with other molecules. Secondary structure predictions have been dealt with in a number of academic publications. Moult, 1996, Curr. Op. Biotech. 7:422-427]; See Chou et al. , 1974, Biochemistry 13:222-245]; Chou et al ., 1974, Biochemistry 113:211-222; See Chou et al. , 1978, Adv. Enzymol. Relat. Areas Mol. Biol. 47:45-148]; See Chou et al. , 1979, Ann. Rev. Biochem. 47:251-276 and Chou et al. , Biophys. J. 26:367-384. Moreover, computer programs are currently available to help predict the secondary structure. For example, two polypeptides or proteins with greater than 30% sequence identity or greater than 40% similarity often have similar structural topologies. The recent increase in the Protein Structure Database (PDB) has provided improved predictability of secondary structures, including the number of potential folds within the structure of a polypeptide or protein. See Holm et al. , 1999, Nucl. Acid. Res. 27:244-247. It has been suggested that a given polypeptide or protein has a limited number of folding, and once a critical number of structures are degraded, structural predictions can be much more accurate (Brenner et al ., 1997, Curr. Op. Struct. Biol. 7: 369-376).

Additional methods for predicting secondary structures include “threading” (Jones, 1997, Curr. Opin. Struct. Biol. 7:377-87); Sippl et al. , 1996, Structure 4: 15-19]), “profile analysis” (Bowie et al. , 1991, Science 253:164-170); Gribskov et al. , 1990, Meth. Enzym. 183:146-159; Gribskov et al. , 1987, Proc. Nat. Acad. Sci. USA 84:4355-4358]), and “evolutionary linkage” (Holm, (1999) supra, and Brenner supra. , (1997)]). In certain embodiments, variants of the antibody include glycosylation variants, wherein the number and/or type of glycosylation sites have been altered compared to the amino acid sequence of the parent polypeptide. In certain embodiments, variants contain more or fewer N -linked glycosylation sites than native proteins. Alternatively, removal of such sequences by substitution removes existing N -linked carbohydrate chains. Rearrangements of N -linked carbohydrate chains are also provided, wherein one or more N -linked glycosylation sites (typically naturally occurring sites) are removed, and one or more new N -linked sites are created. Additional preferred antibody variants include cysteine variants, wherein one or more cysteine residues are deleted from or substituted with another amino acid (eg, serine) compared to the parent amino acid sequence. Cysteine variants may be useful when the antibody has to be refolded into a biologically active conformation, such as after isolation of an insoluble inclusion body. Cysteine variants generally have fewer cysteine residues than native proteins, and typically have an even number to minimize interactions due to unpaired cysteines.

Desired amino acid substitutions (whether conservative or non-conservative) can be determined by one of skill in the art at the point where such substitutions are desired. In certain embodiments, amino acid substitutions can be used to identify important residues of an antibody for human GIPR, or to increase or decrease the affinity of an antibody for human GIPR described herein.

According to certain embodiments, preferred amino acid substitutions are (1) reduced susceptibility to proteolysis, (2) reduced susceptibility to oxidation, and/or (3) binding affinity to form protein complexes. It is a substitution that alters the degree and/or (4) alters the binding affinity, and/or (5) imparts or alters other physicochemical or functional properties to such polypeptides. According to certain embodiments, single or multiple amino acid substitutions (in certain embodiments, conservative amino acid substitutions) are in a naturally occurring sequence (in certain embodiments, a portion of a polypeptide that is outside the domain(s) forming intermolecular contacts). Can be done. In certain embodiments, conservative amino acid substitutions are typically unable to substantially alter the structural characteristics of the parent sequence (e.g., the replacement amino acid does not destroy the helix present in the parent sequence, or other types characterizing the parent sequence. Should not collapse the secondary structure of). Examples of art-recognized polypeptide secondary and tertiary structures are described in Proteins, Structures and Molecular Principles (Creighton, Ed., WH Freeman and Company, New York (1984)); Introduction to Protein Structure (Branden and Tooze, Eds., Garland Publishing, New York, NY (1991)); And Thornton et al. , 1991, Nature 354:105, each of which is incorporated herein by reference.

In certain embodiments, antibodies of the invention may be chemically bound to polymers, lipids or other moieties.

The antigen binding agent may comprise at least one of the CDRs described herein incorporated into a biocompatible framework structure. In one embodiment, the biocompatible framework structure is a conformationally stable structural support capable of presenting one or more amino acid sequences (e.g., CDRs, variable regions, etc.) that bind to an antigen in a localized surface region. , Or a polypeptide or portions thereof sufficient to form a framework, or scaffold. Such structures may be naturally occurring polypeptides or polypeptide “folds” (structural motifs), or may have one or more modifications such as additions, deletions or substitutions of amino acids relative to the naturally occurring polypeptide or fold. Such scaffolds may be derived from polypeptides of any species (or more than one species) such as humans, other mammals, other vertebrates, invertebrates, plants, bacteria or viruses.

Typically, biocompatible framework structures are based on protein scaffolds or backbones that are not immunoglobulin domains. For example, fibronectin, ankyrin, lipocalin, neocarzinostain, cytochrome b, CP1 zinc finger, PST1, coiled coil, LACI-D1 , Z domains and tendamistat domains can be used (see, eg, Nygren and Uhlen, 1997, Current Opinion in Structural Biology 7:463-469).

Additionally, one of skill in the art will recognize that suitable binding agents include portions of such antibodies, such as one or more of heavy chain CDR1, CDR2, CDR3, light chain CDR1, CDR2 and CDR3 as specifically disclosed herein. At least one of the regions of heavy chain CDR1, CDR2, CDR3, light chain CDR1, CDR2, and CDR3 may have at least one amino acid substitution as long as the antibody retains the binding specificity of the unsubstituted CDR. The non-CDR portion of the antibody may be a non-protein molecule, wherein the binding agent cross-blocks the binding of the antibody disclosed herein to human GIPR and/or inhibits GIP signaling activity through the receptor. The non-CDR portion of the antibody is a non-protein molecule in which the antibody exhibits a binding pattern similar to that exhibited by at least one of antibodies L2H2/L6H5 to human GIPR peptides in a competition binding assay and/or neutralizes the activity of GIP. I can. The non-CDR portion of the antibody may be composed of amino acids, wherein the antibody is a recombinant binding protein or synthetic peptide, and the recombinant binding protein cross-blocks binding of the antibodies disclosed herein to human GIPR and/or in vitro or in vivo. Neutralizes the activity of GIP. The non-CDR portion of the antibody may be composed of amino acids, wherein the antibody is a recombinant antibody and the recombinant antibody exhibits a binding pattern similar to that exhibited by at least one of antibodies L2H2/L6H5 to human GIPR peptides in a competitive binding assay/ Or neutralizes the activity of GIP.

Fusion protein of GIPR antibody and GLP-1 or reverse GLP-1

In one embodiment, herein, an antibody that specifically binds to GIPR and 1, 2, 3, 4, 5, 6, 7, or 8 GLP-1 fragments or reverse GLP-1 fragments A fusion protein of GIPR antibody and GLP-1 is provided, including, wherein the fusion protein connects the carboxy terminus of the GLP-1 fragment to the amino terminus of the light or heavy chain of the GIPR antibody through a peptide linker sequence (Linker), The amino terminus of the reverse GLP-1 fragment is linked to the carboxy terminus of the light or heavy chain of the GIPR antibody.

In another embodiment, the GIPR herein comprising an antibody that specifically binds to GIPR and 1, 2, 3, 4, 5, 6, 7, or 8 GLP-1 fragments. A fusion protein of the antibody and GLP-1 is provided; The fusion protein connects the carboxyl end of the GLP-1 fragment to the amino end of the GIPR antibody light or heavy chain through a peptide linker sequence (Linker), or connects the amino end of the reverse GLP-1 fragment to the carboxy end of the GIPR antibody light or heavy chain. Connect.

In another embodiment, herein comprising an antibody that specifically binds to GIPR and 1, 2, 3, 4, 5, 6, 7, or 8 reverse GLP-1 fragments, A fusion protein of GIPR antibody and GLP-1 is provided; The fusion protein connects the amino terminus of the reverse GLP-1 fragment to the carboxy terminus of the light or heavy chain of the GIPR antibody.

In another embodiment, provided herein is a fusion protein of a GIPR antibody and GLP-1, comprising an antibody that specifically binds to GIPR and one, two, three, or four GLP-1 fragments; The fusion protein connects the carboxyl terminus of the GLP-1 fragment to the amino terminus of the GIPR antibody light chain or heavy chain through a peptide linker sequence (Linker).

In another embodiment, provided herein is a fusion protein of a GIPR antibody and GLP-1, comprising an antibody that specifically binds to GIPR and one, two, three or four reverse GLP-1 fragments; The fusion protein connects the amino terminus of the reverse GLP-1 fragment to the carboxy terminus of the light or heavy chain of the GIPR antibody.

In another embodiment, provided herein is a fusion protein of a GIPR antibody and GLP-1, comprising an antibody that specifically binds to GIPR and two GLP-1 fragments; The fusion protein connects the carboxyl terminus of the GLP-1 fragment to the amino terminus of the GIPR antibody light chain or heavy chain through a peptide linker sequence (Linker).

In another embodiment, provided herein is a fusion protein of a GIPR antibody and GLP-1, comprising an antibody that specifically binds to GIPR and two reverse GLP-1 fragments; The fusion protein connects the amino terminus of the reverse GLP-1 fragment to the carboxy terminus of the light or heavy chain of the GIPR antibody.

In another embodiment, provided herein is a GLP-1 fusion protein comprising a GIPR antibody and two GLP-1 fragments; The fusion protein connects the carboxyl end of the GLP-1 fragment to the amino end of the GIPR antibody light or heavy chain through a peptide linker sequence (Linker) (N'-GLP-1-Linker-R-C'); Linking the carboxy terminus of the GLP-1 fragment to the amino terminus of the heavy chain of the GIPR antibody (N'-GLP-1-Linker-R-C'); Here, N'represents the amino terminal of the fusion protein polypeptide chain, C'represents the carboxy terminal of the fusion protein polypeptide chain, GLP-1 represents the GLP-1 fragment, and R represents the light or heavy chain of the GIPR antibody. Represents an amino acid sequence, and Linker represents a peptide linker sequence.

In another embodiment, provided herein is a GLP-1 fusion protein comprising a GIPR antibody and two reverse GLP-1 fragments; The fusion protein connects the amino terminus of the reverse GLP-1 fragment to the carboxy terminus of the GIPR antibody light or heavy chain (N'-R-Linker-reverse GLP-1-C'); Linking the amino terminus of the reverse GLP-1 fragment to the carboxy terminus of the heavy chain of the GIPR antibody via a peptide linker sequence (Linker) (N'-R-Linker-reverse GLP-1-C'); Where N'represents the amino terminal of the fusion protein polypeptide chain, C'represents the carboxy terminal of the fusion protein polypeptide chain, reverse GLP-1 represents the reverse GLP-1 fragment, and R represents the light or heavy chain of the GIPR antibody. Represents the amino acid sequence of, and Linker represents the peptide linker sequence.

In a further embodiment, provided herein is a GLP-1 fusion protein comprising a GIPR antibody and two GLP-1 fragments; The fusion protein connects the carboxy terminus of the GLP-1 fragment to the amino terminus of the GIPR antibody light chain through a peptide linker sequence (Linker) (N'-GLP-1-Linker-R-C'); Here, N'represents the amino terminal of the fusion protein polypeptide chain, C'represents the carboxy terminal of the fusion protein polypeptide chain, GLP-1 represents the GLP-1 fragment, and R represents the amino acid sequence of the GIPR antibody light chain. And Linker represents a peptide linker sequence.

In one embodiment, in the GLP-1 fusion protein provided herein, the GLP-1 fragment is independently selected from one of the following amino acid sequences: SEQ ID NO: 105, SEQ ID NO: 106, SEQ ID NO: 107, SEQ ID NO: 108, and SEQ ID NO: 109. In one embodiment, in the GLP-1 fusion protein provided herein, the reverse GLP-1 fragment is independently selected from one of the following amino acid sequences: SEQ ID NO: 119, SEQ ID NO: 120, SEQ ID NO: 121, SEQ ID NO: 122, and SEQ ID NO: 123.

In one embodiment, in the GLP-1 fusion protein provided herein, the peptide linker sequence is independently 1 to 200 amino acid residues, 2 to 100 amino acid residues, 5 to 50 amino acid residues, 6 It contains from 10 to 25 amino acid residues, or from 10 to 20 amino acid residues.

In another embodiment, in the GLP-1 fusion protein provided herein, the peptide linker sequence is independently selected from the following amino acid sequences: SEQ ID NO: 110, SEQ ID NO: 111, and SEQ ID NO: 112 .

Nucleic acid

In one aspect, the invention provides an isolated nucleic acid molecule encoding an antibody provided herein. The nucleic acid, for example, encodes all or part of the antibody or GLP-1 fusion protein, for example, one or both chains of the antibody of the invention, or a fragment, derivative, mutein, or variant thereof. Polynucleotide; Enough polynucleotides to be used as hybridization probes; PCR primers or sequencing primers for identifying, analyzing, mutating, or amplifying a polynucleotide encoding a polypeptide; Anti-sense nucleic acids for inhibiting the expression of polynucleotides, and complementary sequences of those described above. The nucleic acid can be of any length. This is, for example, 5 or more, 10 or more, 15 or more, 20 or more, 25 or more, 30 or more, 35 or more, 40 or more, 45 or more, 50 or more, 75 nucleotides in length. More than 100, more than 125, more than 150, more than 175, more than 200, more than 250, more than 300, more than 350, more than 400, more than 450, more than 500, more than 750 , May be 1,000 or more, 1,500 or more, 3,000 or more, 5,000 or more, and/or may contain one or more additional sequences, such as regulatory sequences, and/or be part of a larger nucleic acid, e.g., a vector. I can. Nucleic acids can be single-stranded or double-stranded, and can include RNA and/or DNA nucleotides, and artificial variants thereof (eg, peptide nucleic acids).

Nucleic acids encoding antibody polypeptides (eg, heavy or light chains, only variable domains, or full length) can be isolated from B-cells of mice immunized with GIPR antigens. The nucleic acid of the antibody or GLP-1 fusion protein can be isolated by conventional procedures such as polymerase chain reaction (PCR).

Nucleic acid sequences encoding the variable regions of the heavy and light chains are presented above. Those of skill in the art will recognize that due to the degenerate nature of the genetic code, each of the polypeptide sequences disclosed herein is encoded by a number of different nucleic acid sequences. The present invention provides each degenerate nucleotide sequence encoding each antibody or GLP-1 fusion protein provided herein.

The invention further provides nucleic acids that hybridize under certain hybridization conditions to other nucleic acids (eg, nucleic acids comprising the nucleotide sequence of any of L2H2/L6H5). Methods of hybridizing nucleic acids are well known in the art. See, for example, Current Protocols in Molecular Biology, John Wiley & Sons, NY (1989), 6.3.1-6.3.6. As defined herein, for example, moderately stringent hybridization conditions are a prewash solution containing 5x sodium chloride/sodium citrate (SSC), 0.5% SDS, 1.0 mM EDTA (pH 8.0), of about 50% formamide. Hybridization buffer, 6xSSC, and a hybridization temperature of 55° C. (or other similar hybridization solution such as a solution containing about 50% formamide with a hybridization temperature of 42° C.), and 0.5×SSC, 0.1% SDS. Washing conditions of 60°C are used. Stringent hybridization conditions are hybridization in 6xSSC at 45°C, followed by washing at least once in 0.1xSSC and 0.2% SDS at 68°C. Moreover, those skilled in the art will keep nucleic acids comprising nucleotide sequences that are at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% identical to each other, typically hybridized with each other. The hybridization and/or washing conditions can be manipulated to increase or decrease the stringency of hybridization, if possible. Guidance for devising basic parameters and suitable conditions that influence the choice of hybridization conditions, see, for example, Sambrook, Fritsch, and Maniatis [1989, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring. Harbor, NY, chapters 9 and 11]; And in Current Protocols in Molecular Biology, 1995, Ausubel et al. , Eds., John Wiley & Sons, Inc., sections 2.10 and 6.3-6.4, and can be readily determined by a person skilled in the art based on, for example, the length and/or base composition of the DNA. Alterations can be introduced into the nucleic acid by mutation, thereby resulting in alteration of the amino acid sequence of the polypeptide (eg, antibody) it encodes. Mutations can be introduced using any technique known in the art. In one embodiment, one or more specific amino acid residues are altered, eg, using a site-directed mutagenesis protocol. In another embodiment, one or more randomly selected residues are altered, eg, using a random mutagenesis protocol. Regardless of how alterations are made, the mutant polypeptide can be expressed and screened for desired properties.

Mutations can be introduced into a nucleic acid without significantly altering the biological activity of the polypeptide encoded by the nucleic acid. For example, nucleotide substitutions can result that lead to amino acid substitutions at non-essential amino acid residues. In one embodiment, the nucleotide sequence provided herein for the L1-L11 and H1-H9, or GLP-1 fusion protein, or fragment, variant, or derivative thereof, is such that it produces a sequence with two or more different amino acid residues. Is mutated to encode the amino acid sequences provided herein for LI to L11 and H1 to H9 comprising one or more deletions or substitutions of amino acid residues. In another embodiment, mutagenesis comprises amino acids adjacent to one or more amino acid residues set forth herein for the L1-L11 and H1-H9 or GLP-1 fusion proteins to produce a sequence having two or more different amino acid residues. Insert. Alternatively, one or more mutations can be introduced into a nucleic acid that selectively alters the biological activity (eg, binding to GIPR) of the polypeptide that the nucleic acid encodes. For example, mutations can alter biological activity quantitatively or qualitatively. Examples of quantitative changes include increasing, decreasing or eliminating activity. Examples of qualitative alterations include alteration of the antigen specificity of an antibody or GLP-1 fusion protein.

In another aspect, the invention provides nucleic acid molecules suitable for use as primers or hybridization probes for detection of the nucleic acid sequences of the invention. The nucleic acid molecule of the invention is only a portion of a nucleic acid sequence encoding the full-length polypeptide of the invention, e.g., a fragment that can be used as a probe or primer, or an active portion of the polypeptide of the invention (e.g., GIPR binding moiety) May include a fragment that encodes.

Probes based on the sequence of a nucleic acid of the present invention can be used to detect a nucleic acid or similar nucleic acid, for example a transcript encoding a polypeptide of the present invention. The probe may contain a labeling group, such as a radioactive isotope, a fluorescent compound, an enzyme, or an enzyme cofactor. Such probes can be used to identify cells expressing the polypeptide.

In another aspect, vectors provided herein comprise nucleic acids encoding a polypeptide of the invention or a portion thereof. Examples of vectors include, but are not limited to, plasmids, viral vectors, non-episomal mammalian vectors and expression vectors, such as recombinant expression vectors.

The recombinant expression vectors provided herein may contain a nucleic acid of the present invention in a form suitable for expression of the nucleic acid in a host cell. Recombinant expression vectors contain one or more regulatory sequences selected based on the host cell to be used for expression, which is operatively linked to the nucleic acid sequence to be expressed. Regulatory sequences include regulatory sequences that direct the constitutive expression of nucleotide sequences in many types of host cells (e.g., SV40 early gene enhancer, Rous sarcoma virus promoter and cytomegalovirus promoter), and expression of nucleotide sequences only in certain host cells. Directing regulatory sequences (eg, tissue-specific regulatory sequences (Voss et al. , 1986, Trends Biochem. Sci. 11:287), Maniatis et al. , 1987, Science 236:1237). And each of these disclosures is incorporated herein by reference in its entirety), and regulatory sequences directing the inducible expression of nucleotide sequences in response to specific treatments or conditions (e.g., metallothionein in mammalian cells). Promoters and tet-reactive and/or streptomycin responsive promoters in both prokaryotic and eukaryotic systems (see the same literature). It will be appreciated by those skilled in the art that the design of the expression vector can depend on factors such as the choice of host cells to be transformed, the level of expression of the desired protein, and the like. The expression vector of the present invention can be introduced into a host cell to produce a protein or peptide comprising a fusion protein or peptide encoded by a nucleic acid as described herein.

In another aspect, the present invention provides a host cell into which the recombinant expression vector of the present invention has been introduced. The host cell can be any prokaryotic or eukaryotic cell. Prokaryotic host cells are Gram-negative or Gram-positive organisms, such as E. Coli or bacillus . Higher eukaryotic cells include insect cells, yeast cells, and established cell lines of mammalian origin. Examples of suitable mammalian host cell lines include Chinese hamster ovary (CHO) cells or derivatives thereof such as Veggie CHO and related cell lines growing in serum-free medium (see Rasmussen et al. , 1998, Cytotechnology 28:31) or DHFR. This deficient CHO strain DXB-11 (see Urlaub et al. , 1980, Proc. Natl. Acad. Sci. USA 77:4216-20) is included. Additional CHO cell lines include CHO-K1 (ATCC#CCL-61), EM9 (ATCC# CRL-1861), and W20 (ATCC# CRL-1862). Additional host cells include the COS-7 family of monkey kidney cells (ATCC# CRL-1651) (see Gluzman et al. , 1981, Cell 23:175), L cells, C127 cells, 3T3 cells (ATCC CCL- 163), AM-1/D cells (described in U.S. Patent No.6210924), HeLa cells, BHK (ATCC CRL-10) cell line, CV1/EBNA cell line derived from African green monkey kidney cell line CV1 (ATCC CCL- 70) (see McMahan et al ., 1991, EMBO J. 10:2821), human embryonic kidney cells such as 293, 293 EBNA or MSR 293, human epidermal A431 cells, human Colo205 cells, other transformed primates Cell lines, normal diploid cells, cell strains derived from in vitro cultures of primary tissues, primary explants, HL-60, U937, HaK or Jurkat cells. Cloning and expression vectors suitable for use with bacterial, fungal, yeast, and mammalian cell hosts are described in Pouwels et al., Cloning Vectors: A Laboratory Manual, Elsevier, NY, 1985.

Vector DNA can be introduced into prokaryotic or eukaryotic cells through conventional transformation or transfection techniques. For stable transfection of mammalian cells, it is known that only a small fraction of the cells are capable of incorporating foreign DNA into their genome, depending on the expression vector and transfection technique used. In order to identify and select such an integrant, a gene encoding a selectable marker (eg, for antibiotic resistance) is generally introduced into the host cell along with the gene of interest. Preferred selectable markers include those that confer resistance to drugs such as G418, hygromycin and methotrexate. Cells stably transfected with the introduced nucleic acid can be identified by drug selection, in particular, among several methods (e.g., cells incorporating the selectable marker gene will survive while other cells will die). .

Transformed cells can be cultured under conditions that promote expression of the polypeptide, and the polypeptide is recovered by conventional protein purification procedures. One such purification procedure is described in the Examples below. Polypeptides contemplated for use herein include substantially homogeneous recombinant mammalian GIPR antibodies or GLP-1 fusion protein polypeptides that are substantially free of endogenous contaminants.

GIPR antibody activity

The activity of the GIPR antibody specifically binds to GIPR, inhibits or blocks GIP signaling, and then shows therapeutic biological effects in the treatment of, for example, obesity, T2DM and/or nonalcoholic steatohepatitis (NASH). Refers to the effect of the antibody provided to. The terms "reducing the biological activity of GIP signaling" or "inhibiting or blocking the biological activity of GIP signaling" are used to inhibit or block a downstream cellular response to GIP by binding to GIPR in vivo. It refers to the effect of a GIPR antibody or a fusion protein of GLP-1 therewith. Such reactions include, but are not limited to, insulinotropic effects, promotion of fat accumulation, and inhibition of lipolysis. In one embodiment, a mouse antibody or humanized antibody provided herein specifically binds human GIPR. Such antibodies include antagonistic or neutralizing antibodies that reduce or neutralize GIP signaling.

In one embodiment, the K _d of an antibody provided herein that binds to human GIPR ranges from approximately 0.01 nM to 1000 nM, 0.1 nM to 500 nM, 0.5 nM to 200 nM, 1 nM to 200 nM, or 10 nM to 100 nM. to be. In another embodiment, the K _d of an antibody provided herein that binds to GIPR is approximately 1 nM to 200 nM. In another embodiment, the K _d of an antibody provided herein that binds to GIPR is approximately 10 nM to 100 nM. In another embodiment, the K _d of an antibody provided herein that binds to GIPR is approximately 1 nM, 2 nM, 5 nM, 10 nM, 20 nM, 30 nM, 40 nM, 50 nM, 60 nM, 70 nM, 80 nM, 90 nM, or 100 nM.

In one embodiment, the IC ₅₀ of an antibody provided herein in antagonizing GIP signaling is approximately 0.01 nM to 500 nM, 0.1 nM to 200 nM, 0.5 nM to 200 nM, 1 nM to 200 nM, or 10 nM to In the range of 100 nM. In another embodiment, the IC ₅₀ of an antibody provided herein in antagonizing GIP signaling is approximately 1 nM to 200 nM. In another embodiment, the IC ₅₀ of an antibody provided herein in antagonizing GIP signaling is approximately 10 nM to 100 nM. In another embodiment, the IC ₅₀ of an antibody provided herein in antagonizing GIP signaling is approximately 1 nM, 2 nM, 5 nM, 10 nM, 20 nM, 30 nM, 40 nM, 50 nM, 60 nM, 70 nM, 80 nM, 90 nM, or 100 nM.

In one embodiment, the antibodies provided herein specifically bind to GIPR with one or more of the following properties:

a. The property of providing a Kd substantially similar to the reference antibody in binding to GIPR;

b. The property of providing an IC50 substantially similar to the reference antibody in antagonizing GIPR activated by GIP; And

c. The property of competitive binding with a reference antibody for human GIPR.

In one embodiment, the reference antibody comprises a combination of the light chain variable domain amino acid sequence SEQ ID NO: 66 and the heavy chain variable domain amino acid sequence SEQ ID NO: 76. In another embodiment, the reference antibody is a monoclonal antibody L2H2, L6H5, or L10H8.

As used herein, the term "substantially similar" is comparable to the IC ₅₀ or K _d of the reference antibody or approximately 200%, 180%, 160%, 150%, 140%, 120%, 110%, 100 %, 99%, 98%, 97%, 95%, 90%, 85%, 80%, 75%, 70%, 65%, or 50%. In one embodiment, the reference antibody is, for example, an antibody comprising a combination of heavy chain SEQ ID NO: 76 and light chain SEQ ID NO: 66. In another embodiment, the reference antibody comprises L2H2, L6H5, or L10H8.

Biological activity of fusion protein of GIPR antibody and GLP-1

The biological activity of the fusion protein of GIPR antibody and GLP-1 includes the biological activity of GLP-1 and the activity of GIPR antibody. The activity of the GIPR antibody is as described above. "GLP-1 biological activity" binds to and activates the GLP-1 receptor in vivo and triggers a cellular signaling response, and has a therapeutic effect, such as for obesity, type 2 diabetes, or nonalcoholic steatohepatitis. It refers to the biological activity of the fusion protein of GIPR antibody and GLP-1. Signal transduction responses include, but are not limited to, increased insulin secretion, glucagon secretion, appetite suppression, weight loss, satiety induction, apoptosis inhibition, pancreatic β cell proliferation induction, and pancreatic β cell differentiation. When the biological activities of GLP-1 and GIPR antibodies are combined, the GLP-1 fusion proteins provided herein can be used to treat various diseases and disorders associated with GLP-1R and GIPR. Since fusion proteins exert biological effects by acting on GLP-1R and/or GIPR, GLP-1 fusion protein treatments provided herein will benefit from “increased GLP-1R signaling” or “reduced GIPR signaling”. It can be used to treat a subject having a disease or condition. Such subjects include non-insulin-dependent diabetes mellitus, insulin-dependent diabetes mellitus, stroke (GLP-1R, see WO 00/16797), myocardial infarction (GLP-1R, see WO 98/08531), obesity (GLP-1R, WO 98 /19698; GIPR, Furija et al. , 2008, PLoS ONE 3:e3163; US 2017/0275370 A1), postoperative catabolism changes (GLP-1R, see US 6,006,753), functional indigestion and irritable bowel Syndrome (GLP-1R, see WO99/64060), hepatic steatosis (GIPR, see US 2017/0275370A1), non-alcoholic fatty liver disease (GLP-1R, Debra et al. , 2016, Hepatobiliary Surg Nutr 5: 515-518 ] Reference; GIPR, see US 2017/0275370 A1), non-alcoholic steatohepatitis (GLP-1, see Armstrong et al. , 2013, BMJ Open 3:e003995; see GIPR, US 2017/0275370 A1) Thus referred to as a subject in need of “GLP-1R stimulation therapy” or “in need of reducing GIPR stimulation”, and at risk of developing non-insulin-dependent diabetes (see WO 00/07617), impaired glucose tolerance, or Subjects with impaired fasting blood sugar, subjects weighing about 25% greater than normal height and weight, and subjects who have undergone partial pancreatectomy.

In one embodiment, changes in the biological activity of a GIPR antibody or a fusion protein of GLP-1 with it are detected using a direct cAMP assay that quantifies the function of a GIPR antibody or GLP-1 fusion protein in inhibiting GIPR in vitro. .

Pharmaceutical composition

In one embodiment, a pharmaceutical composition provided herein comprises a GIPR antibody provided herein and one or more pharmaceutically acceptable carriers.

In another embodiment, a pharmaceutical composition provided herein comprises a fusion protein of a GIPR antibody and GLP-1 provided herein, and one or more pharmaceutically acceptable carriers.

The term “carrier” as used herein includes carriers, pharmaceutical excipients, or stabilizers that are harmless to exposure to cells or mammals at the dosages and concentrations employed.

Treatment method

In one embodiment, provided herein is a method of treating, preventing, or ameliorating type 2 diabetes comprising administering to a subject a therapeutically effective dosage of a GIPR antibody provided herein or a pharmaceutical composition thereof.

In another embodiment, provided herein is a method of treating, preventing, or ameliorating a non-alcoholic fatty liver disease comprising administering to the subject a therapeutically effective dose of a GIPR antibody provided herein, or a pharmaceutical composition thereof. do.

In another embodiment, herein, treatment or prevention of non-alcoholic fatty liver disease comprising administering to a subject a fusion protein of a GIPR antibody and GLP-1 provided herein in a therapeutically effective dose, or a pharmaceutical composition thereof. Or, a method of improving is provided.

In another embodiment, provided herein is a method of treating, preventing, or ameliorating non-alcoholic steatohepatitis comprising administering to the subject a therapeutically effective dosage of a GIPR antibody provided herein, or a pharmaceutical composition thereof. do.

In another embodiment, herein, a therapeutically effective dose of a fusion protein of a GIPR antibody and GLP-1 provided herein, or a pharmaceutical composition thereof, is administered to a subject. Or, a method of improving is provided.

In another embodiment, provided herein is a method of treating, preventing, or ameliorating type 2 diabetes comprising administering to the subject a therapeutically effective dose of a GIPR antibody provided herein, or a pharmaceutical composition thereof. do.

In yet another embodiment, the treatment of type 2 diabetes, comprising administering to the subject a therapeutically effective dose of a fusion protein of a GIPR antibody and GLP-1 provided herein, or a pharmaceutical combination thereof, or Methods of preventing or improving are provided.

In another embodiment, provided herein is a method of treating, preventing, or ameliorating obesity comprising administering to the subject a therapeutically effective dosage of a GIPR antibody provided herein, or a pharmaceutical composition thereof.

In a further embodiment, herein a therapeutically effective dose of a fusion protein of a GIPR antibody provided herein and GLP-1, or a pharmaceutical composition thereof, is administered to the subject, treating, preventing, or ameliorating obesity. A method is provided.

In any of the uses provided herein, the pharmaceutical compositions provided herein are for intravenous injection or subcutaneous injection.

As used herein, the term “subject” refers to a mammal, including humans, and is used interchangeably with the term “patient”.

The term “treatment” includes alleviation or prevention of at least one symptom or other aspect of a disorder, or reduction in disease severity. The GIPR antibody provided herein or the fusion protein of GIPR antibody and GLP-1 does not need to provide complete cure or eradicate all symptoms or signs of the disease to be an effective therapeutic agent. As recognized in the art, therapeutic agents can reduce the severity of a given disease state, but need not eliminate all signs of the disease to be effective. Similarly, a prophylactic agent does not have to completely prevent the onset of the disease in order to be effective. (E.g., by reducing the number or severity of symptoms of a disease, increasing the effectiveness of another treatment, or producing another beneficial effect) simply reducing the effect of the disease or developing or exacerbating the disease in a subject It is enough to reduce the likelihood of becoming. One embodiment of the present invention relates to a method comprising administering an antibody to a patient in an amount and for a sufficient time to induce a lasting improvement relative to the baseline of an indicator reflecting the severity of a particular disorder.

The pharmaceutical composition of the GIPR antibody or the fusion protein of the GIPR antibody and GLP-1 can be administered by any suitable technique, including, but not limited to, parenteral administration, topical administration, or administration by inhalation. When injected, the pharmaceutical composition may be administered by bolus injection or continuous infusion, for example via intraarticular, intravenous, intramuscular, intralesional, intraperitoneal or subcutaneous routes. Localized administration at the site of disease or injury, such as transdermal administration and sustained release of implants, is contemplated. Delivery by inhalation includes, for example, nasal or oral inhalation, use of a nebulizer, inhalation of the antibody in the form of an aerosol, and the like. Other alternatives include oral formulations including pills, syrups, or lozenges.

Advantageously, the GIPR antibody or fusion protein of the GIPR antibody provided herein is administered in a composition comprising one or more additional ingredients such as a physiologically acceptable carrier, excipient or diluent. The composition further comprises one or more physiologically active agents as described below. In many specific embodiments, the composition comprises 1, 2, 3, 4, 5, or 6 in addition to one or more antibodies (e.g., murine antibodies or humanized antibodies) or GLP-1 fusion proteins provided herein. Includes physiologically active agents.

In one embodiment, the pharmaceutical composition comprises a murine antibody or humanized antibody or GLP-1 fusion protein provided herein, a buffer suitable for an antibody at a suitable pH, an antioxidant such as ascorbic acid, a low molecular weight polypeptide (e.g., less than 10 amino acids). Polypeptides), proteins, amino acids, carbohydrates such as dextrins, chelating agents such as EDTA, glutathione, stabilizers, and excipients. According to appropriate industry standards, preservatives may also be added. The composition can be formulated as a lyophilizate using a suitable excipient solution as a diluent. Suitable ingredients are non-toxic to recipients at the dosage and concentration used. Further examples of ingredients that can be used in pharmaceutical formulations are described in Remington's Pharmaceutical Sciences, 16th Ed. (1980) and 20th Ed. (2000)]. Mack Publishing Company kits are provided for use by physicians, which include one or more antibodies or GLP-1 fusion proteins of the invention and labels or other instructions for use in treating any of the diseases discussed herein. In one embodiment, the kit comprises a sterile preparation of one or more human antibodies or GLP-1 fusion proteins, which may be in the form of a composition as disclosed above, and may be in one or more vials.

The dosage and frequency of administration may vary depending on factors such as the route of administration, the specific antibody or GLP-1 fusion protein used, the nature and severity of the disease to be treated, whether the disease is acute or chronic, and the size and overall condition of the subject. have. Appropriate dosages can be determined by procedures known in the art, for example, procedures known in clinical trials that may involve dose escalation studies.

The antibody or GLP-1 fusion protein provided herein can be administered, eg, once or more than once, eg, at regular intervals over a period of time. In certain embodiments, the murine antibody or humanized antibody or GLP-1 fusion protein is administered once over a period of at least 1 month or longer, e.g., 1 month, 2 months, or 3 months or even for an indefinite period. do. To treat chronic diseases, long-term treatment is generally most effective. However, for the treatment of acute disease, administration over a shorter period of time, eg, 1 to 6 weeks, may be sufficient. In general, humanized antibodies are administered until the patient shows a medically relevant degree of improvement compared to baseline for the selected indicator or indicators.

Examples of treatment regimens provided herein include an appropriate dose of an antibody or GLP-1 fusion protein, once per week or longer, to treat symptoms caused by type 2 diabetes, obesity, or nonalcoholic steatohepatitis. Includes subcutaneous injection. The antibody or GLP-1 fusion protein can be administered weekly or monthly until the desired result is achieved, for example, until the patient's symptoms subside. Treatment can be resumed if necessary, or alternatively a maintenance dose can be given.

To detect any pressure change, the patient's blood glucose concentration and body weight can be monitored before, during, and/or after treatment with an antibody or GLP-1 fusion protein, such as a human antibody or GLP-1 fusion protein. . For certain diseases, changes in blood sugar may vary depending on factors such as disease progression. Blood sugar levels can be determined using known techniques.

Certain embodiments of the methods and compositions herein include, for example, antibodies or GLP-1 fusion proteins provided herein and one or more GIP antagonists, two or more antibodies or GLP-1 fusion proteins, or antibodies or GLP- 1 involves the use of a fusion protein and one or more other GLP antagonists. In a further embodiment, the antibody or GLP-1 fusion protein is administered alone or in combination with other agents used to treat painful symptoms in the patient. Examples of such agents include protein and non-protein drugs. When several drugs are administered in combination, the dosage should be adjusted according to what is well known in the art. “Co-administration” or combination therapy is not limited to simultaneous administration, but also includes a therapeutic regimen in which the antigen and protein are administered at least once during a course of administration involving administration of at least one other therapeutic agent to a patient.

On the other hand, in the present application, as a method of manufacturing a drug for treating type 2 diabetes, obesity and non-alcoholic steatohepatitis and related disorders, the antibody or GLP-1 fusion protein and pharmaceutical provided herein for the treatment of related diseases A method is provided comprising the step of mixing an acceptable excipient. The pharmaceutical preparation method is as described above.

Further provided herein are compositions, kits, and methods relating to an antibody or GLP-1 fusion protein capable of specifically binding human GIPR. Nucleic acid molecules and derivatives and fragments thereof are also provided, which are polynucleotides encoding all or part of a polypeptide that binds GIPR, e.g., all of a GIPR antibody, antibody fragment, antibody derivative, or GLP-1 fusion protein. Or a nucleic acid encoding a portion. Further provided herein are vectors and plasmids containing such nucleic acids, and cells and cell lines containing such nucleic acids and/or vectors and plasmids. The methods provided herein include, for example, methods of making, identifying, or isolating an antibody or GLP-1 fusion protein that binds to human GIPR, a method of determining whether the antibody or GLP-1 fusion protein binds to GIPR. , And a method of administering an antibody or GLP-1 fusion protein that binds to GIPR to an animal model.

The technical solutions described herein will be further understood by the following examples.

Unless specified, the starting materials and equipment described herein are commercially available, or are those commonly used in the art. Unless otherwise specified, the methods of the following examples are all conventional methods in the art.

1: Preparation of antigen for immunization

CHO-DHFR- cells were seeded in 6-well plates. After 24 hours (hr), cells were transfected with pTM15 plasmid containing the hGIPR gene (see SEQ ID NO: 114 for the nucleotide sequence and SEQ ID NO: 113 for the amino acid sequence). Transfection was performed using Lipofectamine 2000 (Invitrogen) according to the manufacturer's recommended protocol. 48 hours after transfection, the medium was replaced with complete medium containing 300 μg/mL hygromycin, and the medium was changed every 3 days (d). During about 2 weeks of incubation, stable clones were observed. Dispersed cell colonies were removed from the plate and passaged continued until 100% confluence was reached. The constructed stable cell line was analyzed by FACS using a monoclonal antibody against the V5 tag (Life Technologies) to verify positive clones after pressure selection. A large amount of cell-surface hGIPR expression was detected in selected CHO-DHFR-hGIPR cells. Finally, three stable high hGIPR expressing cell lines were identified by subcloning and further validation. These cell lines were used to generate immunogens for antibody production (see Example 2). In addition, in some embodiments, a fusion protein of the extracellular domain of hGIPR and hIgG Fc can also be used as an immunogen for antibody production. The preparation method was as follows: The fusion protein gene of the hGIPR extracellular domain, hIgG Fc and peptide linker was subcloned into the pTM5 plasmid. Cell supernatant was generated by transient mass expression using suspended HEK293 cells, and then hGIPR extracellular domain fusion protein was obtained by affinity chromatography purification.

2: Preparation of antibody

Antibodies against hGIPR can be produced using immunogens comprising any of the following. For example, in certain embodiments, whole cells expressing hGIPR are used as an immunogen to produce antibodies against hGIPR. In addition, in certain embodiments, a fusion protein comprising hFc and the N-terminal extracellular domain of hGIPR is used as an immunogen to generate antibodies against hGIPR. The immunogen and aluminum hydroxide adjuvant were mixed and injected subcutaneously into BALB/c mice (6 weeks to 8 weeks) and boosted once a week. After a total of 6 immunizations, blood samples were taken from the tail vein, serum was separated by centrifugation, and then serum titers were analyzed by FACS. After the highest titer was achieved, the mice were sacrificed and their spleen cells harvested under sterile conditions. The logarithmic growth phase SP2/0 cells were collected, centrifuged, and the cell pellet was resuspended in serum-free culture medium, followed by centrifugation, secondarily resuspended and counted. After mixing the splenocytes and SP2/0 cells at a ratio of 1:1 or more SP2/0 cells:splenocytes, three times of washing-centrifugation were performed. After flicking the pellet from the last centrifugation, 1 mL of pre-warmed PEG-1500 was added dropwise, pipet-mixed for 1 minute, and 30 mL of pre-warmed serum-free medium was slowly added to PEG. The fusion was terminated. The cell pellet was resuspended in the fusion culture medium. 100 μL of splenocytes and feeder layer cells were plated in each well of a 96-well plate. With HAT (sarcine, amethopterin and thymidine) selection to remove unfused cells, fused hybridoma cells and feeder layer cells were co-located in a 96-well plate. -Cultured. After 10 days, the supernatant of hybridoma cells in culture plates was collected for ELISA analysis.

3: ELISA screening of antibodies

CHO-DHFR-hGIPR cells overexpressing hGIPR and CHO-DHFR-blank cells were individually transferred to 96-well plates and allowed to reach 90% confluence. The supernatant of the culture medium was removed, the attached cells were washed twice with PBS, and 100% methanol was added to fix the cells at 4°C. Then, 100 μL of newly made 0.6% H ₂ O ₂ -PBS was added, and after incubation at room temperature for 20 minutes, the cells were washed twice with PBS. After blocking with a 1% BSA solution (dissolved in PBS), the hybridoma supernatant was added and incubated at 4° C. for 90 minutes. After several washings, 100 μL of diluted goat anti-mouse Fc-HRP secondary antibody (Sigma-Aldrich) was added to each well and incubated at 37° C. for 30 minutes. After washing 5 times, 100 μL of TMB chromogenic substrate was added, incubated at 37° C. for 15 minutes, and then 50 μL of 2 MH ₂ SO ₄ was added to terminate the reaction, followed by reading at 450 nm. Further, in certain embodiments, a fusion protein of hFc with the N-terminal extracellular domain of hGIPR is used as the coating antigen. After blocking with 1% BSA (dissolved in PBS), the supernatant of hybridoma cells was added and incubated at 4° C. for 90 minutes. Subsequent steps are the same as the ELISA method above for screening for anti-hGIPR monoclonal antibodies. Positive control is serum from immunized mice; The negative control is the cell culture medium. After preliminary screening by ELISA, several positive hybridoma cell lines secreting hGIPR antibodies were obtained. These hybridoma cell lines secreting hGIPR antibodies were selected and subcloned by limiting dilution. Finally, the supernatant of positive hybridoma cells was verified by FACS analysis (see Example 10).

4: Cloning and subcloning of antibody genes

Hybridoma cells secreting the antibody were collected. Hybridoma mRNA was extracted according to the manufacturer's protocol of the QIAGEN mRNA extraction kit. Then, the extracted mRNA was reverse transcribed into cDNA. The reverse transcription primer was a specific primer for the murine light and heavy chain constant regions, specifically the heavy chain reverse transcription primer was (5'-TTTGGRGGGAAGATGAAGAC-3'), and the light chain reverse transcription primer was (5'-TTAACACTCTCCCCTGTTGAA-3') and (5'- TTAACACTCATTCCTGTTGAA-3'). RT-PCR reaction conditions were as follows: 25°C for 5 minutes, 50°C for 60 minutes, and 70°C for 15 minutes. The reverse transcribed cDNA was diluted to 500 μL with 0.1 mM TE, added to an ultrafiltration centrifuge tube (Amicon Ultra-0.5), and centrifuged at 2,000 g for 10 minutes. The filtrate was removed, 500 μL of 0.1 mM TE was added, and centrifuged at 2,000 g for 10 minutes. The filtrate was removed, and the preparation tube was placed in an inverted state with respect to a new centrifuge tube, and centrifuged at 2,000 g for 10 minutes to obtain purified cDNA. After taking the purified cDNA (10 μL) as a template, 4 μL of 5x tailing buffer (Promega), 4 μL of dATP (1 mM) and 10 U of terminal transferase (Promega) were added, and homogeneous Mixed thoroughly and incubated at 37° C. for 5 minutes, followed by incubation at 65° C. for 5 minutes. The polyA tail cDNA was used as a template and PCR was performed to amplify the light and heavy chain variable region genes of the antibody. All of the upstream primers were oligodT, the heavy chain downstream primers were (5'-TGGACAGGGATCCAGAGTTCC-3') and (5'-TGGACAGGGCTCCATAGTTCC-3'), and the light chain downstream primers were (5'-ACTCGTCCTTGGTCAACGTG-3'). PCR reaction conditions were as follows: 95° C. for 5 minutes; 95°C for 30 seconds, 56°C for 30 seconds, 72°C for 1 minute, 40 cycles; And 7 minutes at 72°C. For sequencing, PCR products were ligated to PMD 18-T vector (Takara Bio). PCR primers were designed based on the DNA sequence of the antibody, and accordingly, the complete light chain, heavy chain signal peptide and variable domain and mouse IgG1 constant region were ligated to the expression vector pTM5.

5: antibody humanization and optimization

First, in order to find the germline gene sequence (Ig Germline Gene sequence) of a human antibody homologous to the mouse antibody variable region sequence for humanization, the sequence of the light and heavy chain variable regions of a mouse antibody is analyzed by NCBI online antibody variable region sequence alignment tool (Ig Blast) was used as an input in the search, and the human gene sequence having the highest homology excluding the CDR sequence was used as a template for CDR grafting to obtain a humanized antibody variable region sequence. The humanized antibody light chain and heavy chain variable region genes were synthesized and combined with a human IgG2 or IgG4 constant region sequence to obtain a full-length recombinant humanized antibody sequence. Recombinant antibodies were expressed according to Example 8, and their affinity for GIPR was analyzed by FACS as described in Example 10 to select the antibody with the best affinity. The variable region sequence of the humanized antibody was engineered by site-directed mutagenesis to further improve the affinity for GIPR.

6: Subcloning of gene of humanized hGIPR antibody

The heavy and light chain variable region gene sequences of the optimized humanized antibody were synthesized by outsourcing. In the process, two restriction sites, 5'-terminal NheI and 3'-terminal SalI, were introduced into the heavy chain variable region sequence. The complete heavy chain variable region was ligated with the heavy chain constant region in the expression vector of pTM5. Similarly, by introducing NheI at the 5'end and BsiwI at the 3'-end, the light chain variable region was ligated with the light chain constant region in the expression vector of pTM5.

7: Construction of fusion protein of humanized hGIPR antibody and GLP-1

The optimized humanized antibody was fused with GLP-1 or its derivative sequence through the N-terminus or C-terminus of the light chain to form a GLP-1 fusion protein, and the two sequences were linked by a bridge, a peptide linker sequence (Linker). . The nucleotide sequence of the signal peptide-GLP-1-Linker was synthesized by Genscript Biotechnology Co., Ltd. Using the synthetic gene as a template, the sequence of the "signal peptide-GLP1-Linker" part was amplified using PCR. Further, the nucleotide sequence of the humanized antibody was used as a template, and the sequence of the antibody of the fusion protein sequence was amplified. Subsequently, through overlapping PCR, the "signal peptide-GLP-1-peptide linker" portion of the nucleic acid sequence of the fusion protein was linked with the antibody portion, and two restriction enzyme sites Nhe1 and Not1 were attached to both ends of the primer. By introduction, the complete fusion protein sequence and the expression vector pTM5 were linked together.

8: Transient expression of the fusion protein of hGIPR antibody and GLP-1

HEK293 or CHO suspension cells (5x10 ⁵ /mL) were inoculated into shaker flasks. After spinning at 37° C. for 24 hours, the cell density reached 1×10 ⁶ /mL, and the cells were used for transfection. Polyethyleneimine (PEI) was used as a transfection reagent, which was mixed with DNA. A mixture of PEI/DNA was added to the cell culture after 15 minutes of incubation. After adding the mixture of PEI/DNA, the cells were continuously cultured at 37° C. and 5% CO ₂ for 24 hours. Then tryptone was added to the cell culture as a supplement for expression. Finally, after protein expression was complete (greater than 96 hours), cell supernatant was collected for antibody purification.

9: Purification and isolation of the fusion protein of hGIPR antibody and GLP-1

After centrifugation (8000 rpm), cells and cell debris were removed from the culture, and the supernatant was filtered through a 0.22 μm filter. The clarified supernatant was used for purification. The purification process was completed via chromatograph. The supernatant first flows through the protein A/G affinity column, while the antibodies in it bind to the A/G protein and remain on the column. The antibody was then eluted from the chromatography column using an elution buffer at a low pH (up to 3.0). The low pH eluent was immediately neutralized with 1 M Tris-HCl. The purified antibody was then dialyzed against PBS or other buffer systems.

10: FACS to verify the binding activity of functional hGIPR antibody

CHO-DHFR-hGIPR cells were detached using PBS containing 10 mM EDTA, 10 ⁵ cells per tube were distributed into 1.5 mL EP tubes, and the supernatant was removed after centrifugation. The negative control sample was resuspended in loading buffer (PBS, 2% FBS). For the positive control, a specific concentration of 200 μL GIPR antibody solution was added to the cells and incubated at room temperature; After incubation, the cells were centrifuged at 1500 rpm to remove the supernatant, washed with FACS loading buffer, and centrifuged again. FITC-labeled goat anti-mouse fluorescent antibody or PE-labeled goat anti-human fluorescent antibody was added at a dilution of 1:50 (200 μL/well) and the cells were resuspended and incubated in the dark for 30 minutes at room temperature. . The supernatant was removed after centrifugation, and the cells were washed with FACS loading buffer, centrifuged again, and resuspended in loading buffer for FACS analysis. Recombinant anti-hGIPR functional antibodies specifically bind to GIPR-expressing CHO-DHFR-GIPR cells. In the experimental results shown in Fig. 1, the gray peak and the dotted peak were negative controls; The solid line peak corresponding to 1 μM of antibody L10H8 shows a significant right shift demonstrating specific binding of L10H8 and CHO-DHFR-GIPR.

11: cAMP assay test of hGIPR antibody or hGIPR antibody/GLP-1 fusion protein for in vitro antagonistic activity of GIPR

CHO-DHFR cells stably expressing human GIPR were seeded in a 96-well cell culture plate at 30,000 cells per well, and placed in a 37°C, 5% CO ₂ incubator overnight. The next day, the supernatant was removed and 45 μL/well of serially diluted antibody or hybridoma supernatant was added. Cells were allowed to stand at room temperature for 30 minutes, then GIP peptide (Phoenix Pharmaceuticals, 50 pM) was added at 45 μL/well. Then, the 96-well plate was prepared at 37° C., 5% CO2. Placed in the incubator for 30 minutes, 10 μL/well of 10% Triton X-100 was added to lyse the cells at room temperature, and the lysate was uniformly mixed with a pipette. A cAMP kit (CisBio) was used to detect cAMP produced in the experiment. Transfer the cell lysate of 10 μL/well to a white 384-well plate, add 5 μL/well of cAMP-d2 diluted 1:20, and finally 5 μL/well of Anti-diluted 1:20 -cAMP-Eu3±cryptate was added, and the plate was incubated at room temperature for 1 hour. The time-resolved fluorescence 665 nm/620 nm signal ratio was read on an Envision 2103 microplate reader and then the IC ₅₀ value was calculated using Prism5.0. Figure 2 shows that L7H6 antagonizes GIPR with IC ₅₀ = 7.6 nM. Figure 3 shows that GLP-1-Linker-L7H6 antagonizes GIPR with IC ₅₀ = 14.9 nM.

12: Reporter gene assay test of hGIPR antibody/GLP-1 fusion protein for in vitro activation of GLP-1R

CHO-DHFR- cells co-expressing hGLP1R and CRE-luciferase were seeded into a 96-well cell culture plate at 20000 cells per well and incubated overnight at 37°C. The next day, the culture supernatant was removed. Cells were washed twice with serum-free medium and residual liquid was also removed. Then, 100 μL of serum-free medium containing serially diluted antibody or GLP-1 is added, and incubated at 37° C. for 4 hours. After stimulation, 100 μL of Bright Glo chemiluminescent substrate (Promega) was added. Finally, cell lysates were transferred to white 96-well plates and luminous intensity recorded on a SpectraMax L microplate reader (Molecular Devices). Figure 4 shows that GLP-1-Linker-L7H6 activates hGLP-1R with EC ₅₀ = 0.04 nM.

13: In vivo efficacy study of hGIPR antibody in C57BL/6 obese mice induced by high fat diet

A 60% high fat diet induced C57BL/6 mouse obesity model (DIO mice) was established. Mice were purchased and fed a normal diet for 1 week, and then a certain number of mice were randomly selected as a normal control group to provide a normal mouse diet, and a high fat diet was provided to the remaining animals. All animals were fed continuously for 8 weeks, and body weight and food intake were evaluated once a week. Thereafter, mice receiving a high fat diet were randomly divided into L10H8 group (10 mg/kg) and model group according to their body weight. The drug was injected subcutaneously every 2 days for 6 weeks. It was not administered to the normal control group, and the model group was given the same amount of blank formulation. Body weight, food intake and behavioral observation data were collected during the experiment. On the last day of the experiment, after fasting for 12 hours (access to water is free), orbital blood of animals was collected to separate serum, followed by euthanasia, and the liver was dissected and weighed, and the liver morphology was observed. Serum was tested for ALT, AST, GLU, TC and TG, and liver was tested for TG (see the results in Table 3).

[Table 3]

Biochemical test results of each group after drug administration

After 6 weeks of L10H8 administration, the weight gain of the L10H8 group was only slightly lower than that of the model group, but the liver weight was significantly lower than that of the model group and was close to the normal control group. The liver TG of the L10H8 group was significantly lower than that of the model group, and the serum TG was significantly higher than that of the model group. These results show that L10H8 significantly slows the absorption and accumulation of liver fat. 5 shows the percentage change in body weight for each group. Table 3 summarizes the liver assay data in each group after giving the L10H8 antibody for 6 weeks.

14: Pharmacokinetic study of GIPR antibody/GLP-1 fusion protein in cynomolgus monkey

A total of 6 cynomolgus monkeys (3 males and 3 females) were injected with a single subcutaneous injection of the GLP-1/hGIPR antibody fusion protein at a dose of 2 mg/kg, and before administration through the same body-side forelimb vein (0 minutes), 2 hours, 4 hours, 8 hours, 12 hours, 24 hours, 2 days, 4 days, 6 days, 8 days, 10 days, 12 days, 18 days, 28 days after administration Each 0.6 mL of whole blood sample was collected, and after spontaneous coagulation, it was placed in a centrifuge tube on ice, and then the blood sample was centrifuged to separate the serum, and stored at low temperature (-80°C) until use. The GLP-1 portion and the hGIPR antibody portion of the GLP-1/hGIPR antibody fusion protein in serum samples were individually quantified by ELISA, and the half-life of both in cynomolgus monkeys was determined through software analysis.

15: In vivo efficacy study of hGIPR antibody/GLP-1 fusion protein in cynomolgus monkeys induced by high fat diet

An obese cynomolgus monkey model (DIO cynomolgus monkey) was established with a cynomolgus monkey induced on a 60% high fat diet, and then used to evaluate the in vivo efficacy of GLP-1-Linker-L7H6 via subcutaneous administration. . Monkeys with high fat diet induced were randomly divided into GLP-1-Linker-L7H6 (10 mg/kg) group and model group according to their body weight. Drugs were injected subcutaneously every 2 days for a total of 8 weeks, and the model group was given the same volume of blank formulation. Body weight, food intake and behavioral observation data were collected during the experiment. After the experiment was completed, the animals were euthanized, the liver was dissected and weighed, and the liver morphology was observed. Serum was tested for ALT, AST, GLU, TC and TG, and liver was tested for TG.

The above embodiments are intended to sufficiently disclose and describe to those skilled in the art how to achieve and use the claimed embodiments, and are not intended to limit the scope of the present disclosure. Variations apparent to those skilled in the art are within the scope of the claims herein. All publications, patents, and patent applications cited herein are incorporated herein by reference, as if each of them were specifically and independently incorporated herein by reference.

SEQUENCE LISTING <110> Gmax Biopharm LLC. <120> GIPR Antibody and its Fusion Protein with GLP-1, and Pharmaceutical Composition and Application Thereof <130> 2018 <160> 124 <170> PatentIn version 3.5 <210> 1 <211> 11 <212> PRT <213> Mus musculus <400> 1 Lys Ala Ser Gln Asp Val Gly Thr Ala Val Ala 1 5 10 <210> 2 <211> 7 <212> PRT <213> Mus musculus <400> 2 Trp Ala Tyr Ile Arg His Thr 1 5 <210> 3 <211> 9 <212> PRT <213> Mus musculus <400> 3 Gln Gln Tyr Ser Ser Tyr Pro Trp Thr 1 5 <210> 4 <211> 15 <212> PRT <213> Mus musculus <400> 4 Arg Pro Ser Glu Ser Val Asp Ser Tyr Gly Asn Ser Phe Met His 1 5 10 15 <210> 5 <211> 7 <212> PRT <213> Mus musculus <400> 5 Leu Ala Ser Asn Leu Glu Ser 1 5 <210> 6 <211> 9 <212> PRT <213> Mus musculus <400> 6 Gln Gln Asn Asn Glu Asp Pro Arg Thr 1 5 <210> 7 <211> 11 <212> PRT <213> Mus musculus <400> 7 Lys Ala Ser Glu Asp Ile Tyr Asn Arg Phe Ala 1 5 10 <210> 8 <211> 7 <212> PRT <213> Mus musculus <400> 8 Asp Ala Thr Ser Leu Glu Thr 1 5 <210> 9 <211> 9 <212> PRT <213> Mus musculus <400> 9 Gln Gln Tyr Trp Ser Ile Pro Trp Thr 1 5 <210> 10 <211> 15 <212> PRT <213> Mus musculus <400> 10 Arg Ala Ser Gln Ser Val Asn Thr Ser Val Tyr Ser Tyr Ile His 1 5 10 15 <210> 11 <211> 7 <212> PRT <213> Mus musculus <400> 11 Tyr Ala Ser Asn Leu Glu Ser 1 5 <210> 12 <211> 9 <212> PRT <213> Mus musculus <400> 12 Gln His Ser Trp Asp Phe Pro Tyr Thr 1 5 <210> 13 <211> 15 <212> PRT <213> Mus musculus <400> 13 Arg Ala Ser Gln Ser Val Asn Thr Ala Val Tyr Ser Tyr Ile His 1 5 10 15 <210> 14 <211> 9 <212> PRT <213> Mus musculus <400> 14 Gln His Ser Phe Asp Phe Pro Tyr Thr 1 5 <210> 15 <211> 11 <212> PRT <213> Mus musculus <400> 15 Lys Ala Ser Gln Asp Ile Asn Ser Tyr Leu Ser 1 5 10 <210> 16 <211> 6 <212> PRT <213> Mus musculus <400> 16 Ala Asn Arg Leu Val Asp 1 5 <210> 17 <211> 9 <212> PRT <213> Mus musculus <400> 17 Leu Gln Tyr Asp Glu Phe Pro Phe Thr 1 5 <210> 18 <211> 10 <212> PRT <213> Mus musculus <400> 18 Gly Phe Thr Phe Ser Ser Tyr Ala Met Ser 1 5 10 <210> 19 <211> 16 <212> PRT <213> Mus musculus <400> 19 Ser Ile Ser Ser Gly Gly Ala Thr Tyr Tyr Pro Asp Ser Val Lys Gly 1 5 10 15 <210> 20 <211> 13 <212> PRT <213> Mus musculus <400> 20 Gly Glu Gly Gly Ser Ser Tyr Pro Ala Trp Phe Ala Phe 1 5 10 <210> 21 <211> 17 <212> PRT <213> Mus musculus <400> 21 Glu Ile Ser Ser Gly Gly Ser Tyr Thr Tyr Tyr Pro Asp Thr Val Thr 1 5 10 15 Gly <210> 22 <211> 17 <212> PRT <213> Mus musculus <400> 22 Asp Lys Ala Thr Arg Thr Gly Met Gly Phe Phe Tyr His Thr Met Asp 1 5 10 15 Tyr <210> 23 <211> 10 <212> PRT <213> Mus musculus <400> 23 Gly Tyr Thr Phe Ser Arg Tyr Trp Ile Glu 1 5 10 <210> 24 <211> 16 <212> PRT <213> Mus musculus <400> 24 Glu Ile Leu Pro Gly Ser Asp Ser Pro Asn Tyr Asn Glu Lys Phe Lys 1 5 10 15 <210> 25 <211> 9 <212> PRT <213> Mus musculus <400> 25 Thr Val Val Ala Thr Arg Phe Ala Tyr 1 5 <210> 26 <211> 11 <212> PRT <213> Mus musculus <400> 26 Gly Tyr Ser Ile Thr Ser Asp Tyr Ala Trp Asn 1 5 10 <210> 27 <211> 16 <212> PRT <213> Mus musculus <400> 27 Tyr Ile Ser Tyr Arg Gly Ile Ala Thr Tyr Lys Pro Ser Leu Lys Ser 1 5 10 15 <210> 28 <211> 10 <212> PRT <213> Mus musculus <400> 28 Gly Glu Tyr Gly Pro Gly Asn Phe Asp Phe 1 5 10 <210> 29 <211> 16 <212> PRT <213> Mus musculus <400> 29 Tyr Met Ser Tyr Arg Gly Thr Ala Thr Tyr Asn Pro Phe Leu Lys Ser 1 5 10 15 <210> 30 <211> 10 <212> PRT <213> Mus musculus <400> 30 Tyr Asp Tyr Asp Val Pro Arg Phe Pro Tyr 1 5 10 <210> 31 <211> 33 <212> DNA <213> Mus musculus <400> 31 aaggccagtc aggatgtggg tactgctgta gcc 33 <210> 32 <211> 21 <212> DNA <213> Mus musculus <400> 32 tgggcataca tccggcacac t 21 <210> 33 <211> 27 <212> DNA <213> Mus musculus <400> 33 cagcaatata gcagctatcc gtggacg 27 <210> 34 <211> 45 <212> DNA <213> Mus musculus <400> 34 agacccagtg aaagtgttga tagttatggc aatagtttta tgcac 45 <210> 35 <211> 21 <212> DNA <213> Mus musculus <400> 35 cttgcatcca acctagaatc t 21 <210> 36 <211> 27 <212> DNA <213> Mus musculus <400> 36 cagcaaaata atgaggatcc tcggacg 27 <210> 37 <211> 33 <212> DNA <213> Mus musculus <400> 37 aaggcaagtg aggacatata taatcggttc gcc 33 <210> 38 <211> 21 <212> DNA <213> Mus musculus <400> 38 gatgcaacca gtttggaaac t 21 <210> 39 <211> 27 <212> DNA <213> Mus musculus <400> 39 caacagtatt ggagtattcc gtggacg 27 <210> 40 <211> 45 <212> DNA <213> Mus musculus <400> 40 agggccagcc aaagtgtcaa tacatctgtc tatagttata tacac 45 <210> 41 <211> 21 <212> DNA <213> Mus musculus <400> 41 tatgcatcca acctagaatc t 21 <210> 42 <211> 27 <212> DNA <213> Mus musculus <400> 42 caacacagtt gggattttcc ttacacg 27 <210> 43 <211> 45 <212> DNA <213> Mus musculus <400> 43 agagccagcc agtccgtgaa cacagccgtg tactcttata tccac 45 <210> 44 <211> 27 <212> DNA <213> Mus musculus <400> 44 cagcacagct tcgatttccc ctacacc 27 <210> 45 <211> 33 <212> DNA <213> Mus musculus <400> 45 aaggcgagtc aggacattaa tagctattta agc 33 <210> 46 <211> 18 <212> DNA <213> Mus musculus <400> 46 gcaaacagat tggtagat 18 <210> 47 <211> 27 <212> DNA <213> Mus musculus <400> 47 ctacagtatg atgagtttcc attcacg 27 <210> 48 <211> 30 <212> DNA <213> Mus musculus <400> 48 ggattcactt tcagtagcta tgccatgtct 30 <210> 49 <211> 45 <212> DNA <213> Mus musculus <400> 49 tccattagta gtggtggtgc cacctactat ccagacagtg tgaag 45 <210> 50 <211> 39 <212> DNA <213> Mus musculus <400> 50 ggcgagggcg gtagtagcta cccggcctgg tttgctttc 39 <210> 51 <211> 51 <212> DNA <213> Mus musculus <400> 51 gaaattagta gtggtggtag ttacacctac tatccagaca ctgtgacggg c 51 <210> 52 <211> 51 <212> DNA <213> Mus musculus <400> 52 gataaggcga ctcgaactgg catgggattt ttttaccata ctatggacta c 51 <210> 53 <211> 30 <212> DNA <213> Mus musculus <400> 53 ggctacacat tcagtaggta ctggatagag 30 <210> 54 <211> 51 <212> DNA <213> Mus musculus <400> 54 gagattttac ctggaagtga tagtcctaac tacaatgaga agttcaaggg c 51 <210> 55 <211> 27 <212> DNA <213> Mus musculus <400> 55 acggtagtag ctacaaggtt tgcttac 27 <210> 56 <211> 33 <212> DNA <213> Mus musculus <400> 56 ggctactcaa tcaccagtga ttatgcctgg aac 33 <210> 57 <211> 48 <212> DNA <213> Mus musculus <400> 57 tacataagct acagaggcat cgctacctat aaaccatctc tcaaaagt 48 <210> 58 <211> 30 <212> DNA <213> Mus musculus <400> 58 ggggaatacg gccccggcaa ctttgacttc 30 <210> 59 <211> 48 <212> DNA <213> Mus musculus <400> 59 tacatgagct accgtggtac cgcaacgtac aatccatttc tcaaaagt 48 <210> 60 <211> 30 <212> DNA <213> Mus musculus <400> 60 tatgattacg acgttccccg gtttccttac 30 <210> 61 <211> 108 <212> PRT <213> Mus musculus <400> 61 Asp Ile Val Met Thr Gln Ser His Lys Phe Met Ser Thr Ser Val Gly 1 5 10 15 Asp Arg Val Asn Ile Thr Cys Lys Ala Ser Gln Asp Val Gly Thr Ala 20 25 30 Val Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Lys Leu Leu Ile 35 40 45 Tyr Trp Ala Tyr Ile Arg His Thr Gly Val Pro Asp Arg Phe Thr Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Asn Val Gln Ser 65 70 75 80 Glu Asp Leu Thr Asp Tyr Phe Cys Gln Gln Tyr Ser Ser Tyr Pro Trp 85 90 95 Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg 100 105 <210> 62 <211> 112 <212> PRT <213> Mus musculus <400> 62 Asn Ile Val Leu Thr Gln Ser Pro Ala Ser Leu Ala Val Ser Leu Gly 1 5 10 15 Gln Arg Ala Thr Ile Ser Cys Arg Pro Ser Glu Ser Val Asp Ser Tyr 20 25 30 Gly Asn Ser Phe Met His Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro 35 40 45 Lys Leu Leu Ile Tyr Leu Ala Ser Asn Leu Glu Ser Gly Val Pro Ala 50 55 60 Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Asp 65 70 75 80 Pro Val Glu Ala Asp Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Asn Asn 85 90 95 Glu Asp Pro Arg Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg 100 105 110 <210> 63 <211> 112 <212> PRT <213> Artificial Sequence <220> <223> Synthetic construct <400> 63 Asn Ile Val Leu Thr Gln Ser Pro Ala Ser Leu Ala Val Ser Pro Gly 1 5 10 15 Gln Arg Ala Thr Ile Thr Cys Arg Pro Ser Glu Ser Val Asp Ser Tyr 20 25 30 Gly Asn Ser Phe Met His Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro 35 40 45 Lys Leu Leu Ile Tyr Leu Ala Ser Asn Leu Glu Ser Gly Val Pro Ala 50 55 60 Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Asn 65 70 75 80 Pro Val Glu Ala Asn Asp Thr Ala Asn Tyr Tyr Cys Gln Gln Asn Asn 85 90 95 Glu Asp Pro Arg Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg 100 105 110 <210> 64 <211> 112 <212> PRT <213> Artificial Sequence <220> <223> Synthetic construct <400> 64 Asn Ile Val Leu Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly 1 5 10 15 Glu Arg Ala Thr Ile Asn Cys Arg Pro Ser Glu Ser Val Asp Ser Tyr 20 25 30 Gly Asn Ser Phe Met His Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro 35 40 45 Lys Leu Leu Ile Tyr Leu Ala Ser Asn Leu Glu Ser Gly Val Pro Asp 50 55 60 Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser 65 70 75 80 Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Gln Gln Asn Asn 85 90 95 Glu Asp Pro Arg Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg 100 105 110 <210> 65 <211> 108 <212> PRT <213> Mus musculus <400> 65 Asp Ile Gln Met Thr Gln Ser Ser Ser Ser Phe Ser Val Ser Leu Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Glu Asp Ile Tyr Asn Arg 20 25 30 Phe Ala Trp Phe Gln Gln Lys Pro Gly Asn Ala Pro Arg Leu Leu Ile 35 40 45 Ser Asp Ala Thr Ser Leu Glu Thr Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Lys Asp Tyr Thr Leu Ser Ile Thr Ser Leu Gln Thr 65 70 75 80 Glu Asp Val Ala Thr Tyr Tyr Cys Gln Gln Tyr Trp Ser Ile Pro Trp 85 90 95 Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg 100 105 <210> 66 <211> 112 <212> PRT <213> Mus musculus <400> 66 Asp Ile Val Leu Thr Gln Ser Pro Ala Ser Leu Ala Val Ser Leu Gly 1 5 10 15 Gln Arg Gly Thr Ile Ser Cys Arg Ala Ser Gln Ser Val Asn Thr Ser 20 25 30 Val Tyr Ser Tyr Ile His Trp Tyr Arg Gln Lys Pro Gly Gln Pro Pro 35 40 45 Lys Leu Leu Ile Lys Tyr Ala Ser Asn Leu Glu Ser Gly Val Pro Ala 50 55 60 Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Asn Ile His 65 70 75 80 Pro Val Glu Gly Glu Asp Ser Ala Thr Tyr Tyr Cys Gln His Ser Trp 85 90 95 Asp Phe Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg 100 105 110 <210> 67 <211> 112 <212> PRT <213> Artificial Sequence <220> <223> Synthetic construct <400> 67 Asp Ile Val Leu Thr Gln Ser Pro Ala Ser Leu Ala Val Ser Pro Gly 1 5 10 15 Gln Arg Ala Thr Ile Thr Cys Arg Ala Ser Gln Ser Val Asn Thr Ala 20 25 30 Val Tyr Ser Tyr Ile His Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro 35 40 45 Lys Leu Leu Ile Lys Tyr Ala Ser Asn Leu Glu Ser Gly Val Pro Ala 50 55 60 Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Asn 65 70 75 80 Pro Val Glu Ala Asn Asp Ala Ala Asn Tyr Tyr Cys Gln His Ser Phe 85 90 95 Asp Phe Pro Tyr Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg 100 105 110 <210> 68 <211> 112 <212> PRT <213> Artificial Sequence <220> <223> Synthetic construct <400> 68 Asp Ile Val Leu Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly 1 5 10 15 Glu Arg Ala Thr Ile Asn Cys Arg Ala Ser Gln Ser Val Asn Thr Ser 20 25 30 Val Tyr Ser Tyr Ile His Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro 35 40 45 Lys Leu Leu Ile Lys Tyr Ala Ser Asn Leu Glu Ser Gly Val Pro Asp 50 55 60 Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser 65 70 75 80 Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Gln His Ser Trp 85 90 95 Asp Phe Pro Tyr Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg 100 105 110 <210> 69 <211> 108 <212> PRT <213> Mus musculus <400> 69 Asp Ile Lys Met Thr Gln Ser Pro Ser Ser Val Tyr Ala Ser Leu Gly 1 5 10 15 Glu Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Asp Ile Asn Ser Tyr 20 25 30 Leu Ser Trp Phe Gln Gln Lys Pro Gly Lys Ser Pro Lys Thr Leu Ile 35 40 45 Tyr Arg Ala Asn Arg Leu Val Asp Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Gln Asp Tyr Ser Leu Thr Ile Ser Ser Leu Glu Tyr 65 70 75 80 Glu Asp Met Gly Ile Tyr Tyr Cys Leu Gln Tyr Asp Glu Phe Pro Phe 85 90 95 Thr Phe Gly Ser Gly Thr Lys Leu Glu Met Lys Arg 100 105 <210> 70 <211> 110 <212> PRT <213> Homo sapiens <400> 70 Ser Tyr Val Leu Thr Gln Pro Pro Ser Ala Ser Gly Thr Pro Gly Gln 1 5 10 15 Arg Val Ala Ile Ser Cys Ser Gly Ser Asn Ser Asn Ile Gly Ser Asn 20 25 30 Thr Val His Trp Tyr Gln Gln Leu Pro Gly Ala Ala Pro Lys Leu Leu 35 40 45 Ile Tyr Ser Asn Asn Gln Arg Pro Ser Gly Val Pro Asp Arg Phe Ser 50 55 60 Gly Ser Asn Ser Gly Thr Ser Ala Ser Leu Ala Ile Ser Arg Leu Gln 65 70 75 80 Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Ala Ala Trp Asp Asp Ser Leu 85 90 95 Asn Gly Val Val Phe Gly Gly Gly Thr Lys Val Thr Val Leu 100 105 110 <210> 71 <211> 110 <212> PRT <213> Homo sapiens <400> 71 Ser Tyr Val Leu Thr Gln Pro Pro Ser Ala Ser Gly Thr Pro Gly Gln 1 5 10 15 Arg Val Ala Ile Ser Cys Ser Gly Ser Asn Ser Asn Ile Gly Ser Asn 20 25 30 Thr Val His Trp Tyr Gln Gln Leu Pro Gly Ala Ala Pro Lys Leu Leu 35 40 45 Ile Tyr Gly Asn Asn Asp Arg Pro Ser Gly Val Pro Asp Arg Phe Ser 50 55 60 Gly Ser Asn Ser Gly Thr Ser Ala Ser Leu Ala Ile Ser Arg Leu Gln 65 70 75 80 Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Ala Ala Trp Asp Asp Ser Leu 85 90 95 Asn Gly Val Val Phe Gly Gly Gly Thr Lys Val Thr Val Leu 100 105 110 <210> 72 <211> 121 <212> PRT <213> Mus musculus <400> 72 Glu Val Lys Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 1 5 10 15 Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Ala Met Ser Trp Val Arg Leu Thr Pro Glu Lys Arg Leu Glu Trp Val 35 40 45 Ala Ser Ile Ser Ser Gly Gly Ala Thr Tyr Tyr Pro Asp Ser Val Lys 50 55 60 Gly Arg Phe Thr Phe Ser Arg Asp Asn Ala Arg Asn Ile Leu Tyr Leu 65 70 75 80 Gln Met Asn Ser Leu Arg Ser Glu Asp Thr Ala Met Tyr Tyr Cys Thr 85 90 95 Arg Gly Glu Gly Gly Ser Ser Tyr Pro Ala Trp Phe Ala Phe Trp Gly 100 105 110 Gln Gly Thr Leu Val Thr Val Ser Ala 115 120 <210> 73 <211> 126 <212> PRT <213> Mus musculus <400> 73 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 1 5 10 15 Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Ala Met Ser Trp Val Arg Gln Ser Pro Glu Lys Arg Leu Glu Trp Val 35 40 45 Ala Glu Ile Ser Ser Gly Gly Ser Tyr Thr Tyr Tyr Pro Asp Thr Val 50 55 60 Thr Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr 65 70 75 80 Leu Glu Met Ser Ser Leu Arg Ser Glu Asp Thr Ala Met Tyr Tyr Cys 85 90 95 Val Arg Asp Lys Ala Thr Arg Thr Gly Met Gly Phe Phe Tyr His Thr 100 105 110 Met Asp Tyr Trp Gly Gln Gly Thr Ser Val Thr Val Ser Ser 115 120 125 <210> 74 <211> 126 <212> PRT <213> Artificial Sequence <220> <223> Synthetic construct <400> 74 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Glu Ile Ser Ser Gly Gly Ser Tyr Thr Tyr Tyr Pro Asp Thr Val 50 55 60 Thr Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Val Arg Asp Lys Ala Thr Arg Thr Gly Met Gly Phe Phe Tyr His Thr 100 105 110 Met Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 125 <210> 75 <211> 118 <212> PRT <213> Mus musculus <400> 75 Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Met Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Ile Ser Cys Lys Ala Thr Gly Tyr Thr Phe Ser Arg Tyr 20 25 30 Trp Ile Glu Trp Ile Lys Gln Arg Pro Gly His Gly Leu Glu Trp Ile 35 40 45 Gly Glu Ile Leu Pro Gly Ser Asp Ser Pro Asn Tyr Asn Glu Lys Phe 50 55 60 Lys Gly Lys Ala Thr Phe Thr Ala Asn Thr Ser Ser Asn Thr Ala Tyr 65 70 75 80 Met Gln Leu Ser Ser Leu Thr Phe Glu Asp Ser Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Thr Val Val Ala Thr Arg Phe Ala Tyr Trp Gly Gln Gly Thr 100 105 110 Leu Val Thr Val Phe Ala 115 <210> 76 <211> 119 <212> PRT <213> Mus musculus <400> 76 Asp Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln 1 5 10 15 Ser Leu Ser Leu Thr Cys Thr Val Thr Gly Tyr Ser Ile Thr Ser Asp 20 25 30 Tyr Ala Trp Asn Trp Ile Arg Gln Phe Pro Gly Asn Lys Val Glu Trp 35 40 45 Met Gly Tyr Ile Ser Tyr Arg Gly Ile Ala Thr Tyr Lys Pro Ser Leu 50 55 60 Lys Ser Arg Ile Ser Ile Thr Arg Asp Thr Ser Lys Asn Gln Phe Phe 65 70 75 80 Leu Asn Phe Asn Ser Val Thr Thr Glu Asp Thr Ala Thr Tyr Tyr Cys 85 90 95 Val Arg Gly Glu Tyr Gly Pro Gly Asn Phe Asp Phe Trp Gly Gln Gly 100 105 110 Thr Thr Leu Thr Val Ser Ser 115 <210> 77 <211> 119 <212> PRT <213> Artificial Sequence <220> <223> Synthetic construct <400> 77 Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln 1 5 10 15 Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Tyr Ser Ile Thr Ser Asp 20 25 30 Tyr Ala Trp Asn Trp Ile Arg Gln Pro Pro Gly Lys Gly Val Glu Trp 35 40 45 Met Gly Tyr Ile Ser Tyr Arg Gly Ile Ala Thr Tyr Lys Pro Ser Leu 50 55 60 Lys Ser Arg Ile Thr Ile Ser Arg Asp Thr Ser Lys Asn Gln Phe Ser 65 70 75 80 Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Val Arg Gly Glu Tyr Gly Pro Gly Asn Phe Asp Phe Trp Gly Gln Gly 100 105 110 Thr Thr Val Thr Val Ser Ser 115 <210> 78 <211> 119 <212> PRT <213> Mus musculus <400> 78 Asp Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln 1 5 10 15 Ser Leu Ser Leu Thr Cys Thr Val Thr Gly Tyr Ser Ile Thr Ser Asp 20 25 30 Tyr Ala Trp Asn Trp Ile Arg Gln Phe Pro Gly Asn Lys Leu Glu Trp 35 40 45 Met Gly Tyr Met Ser Tyr Arg Gly Thr Ala Thr Tyr Asn Pro Phe Leu 50 55 60 Lys Ser Arg Ile Ser Ile Asn Arg Asp Thr Ser Lys Asn Gln Val Phe 65 70 75 80 Leu Lys Leu Asn Ser Val Thr Thr Glu Asp Thr Ala Thr Tyr Tyr Cys 85 90 95 Ala Ser Tyr Asp Tyr Asp Val Pro Arg Phe Pro Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Ile Val Ser Ala 115 <210> 79 <211> 119 <212> PRT <213> Homo sapiens <400> 79 Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Gly Thr Phe Ser Ser Tyr 20 25 30 Ala Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Gly Ile Ile Pro Thr Phe Gly Thr Ala Asn Tyr Ala Gln Lys Phe 50 55 60 Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Gln Gly Pro Ile Val Gly Ala Pro Thr Asp Tyr Trp Gly Lys Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 80 <211> 119 <212> PRT <213> Homo sapiens <400> 80 Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Gly Thr Phe Lys Ser Tyr 20 25 30 Ala Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Gly Ile Ile Pro Thr Phe Gly Thr Ala Asn Tyr Ala Gln Lys Phe 50 55 60 Gln Gly Arg Val Thr Ile Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Gln Gly Pro Ile Val Gly Ala Pro Thr Asp Tyr Trp Gly Lys Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 81 <211> 324 <212> DNA <213> Mus musculus <400> 81 gacattgtga tgacccagtc tcacaaattc atgtccacat cagtaggaga cagggtcaac 60 atcacctgca aggccagtca ggatgtgggt actgctgtag cctggtatca acagaaacca 120 gggcaatctc ctaaactact gatttactgg gcatacatcc ggcacactgg agtccctgat 180 cgcttcacag gcagtggatc tgggacagat ttcactctca ccattagcaa tgtgcagtct 240 gaagacttga cagattattt ctgtcagcaa tatagcagct atccgtggac gttcggtgga 300 ggcaccaagc tggaaatcaa acgg 324 <210> 82 <211> 336 <212> DNA <213> Mus musculus <400> 82 aacattgtgc tgacccaatc tccagcttct ttggctgtgt ctctagggca gagggccacc 60 atatcctgca gacccagtga aagtgttgat agttatggca atagttttat gcactggtac 120 cagcagaaac caggacagcc acccaaactc ctcatctatc ttgcatccaa cctagaatct 180 ggggtccctg ccaggttcag tggcagtggg tctgggacag acttcaccct caccattgat 240 cctgtggagg ctgatgatgc tgcaacctat tactgtcagc aaaataatga ggatcctcgg 300 acgttcggtg gaggcaccaa gctggaaatc aaacgg 336 <210> 83 <211> 336 <212> DNA <213> Artificial Sequence <220> <223> Synthetic construct <400> 83 aatatcgtgc tgacccagtc tccagccagc ctggccgtgt ctccaggaca gagggccacc 60 atcacatgca gacctagcga gtccgtggat tcctacggca actctttcat gcactggtat 120 cagcagaagc ccggccagcc ccctaagctg ctgatctacc tggccagcaa tctggagtcc 180 ggagtgcctg caaggttctc tggaagcgga tccggaaccg actttaccct gacaatcaac 240 ccagtggagg ccaacgatac agccaattac tattgtcagc agaacaatga ggacccacgg 300 acctttggcg gaggaacaaa ggtggagatc aagcgt 336 <210> 84 <211> 336 <212> DNA <213> Artificial Sequence <220> <223> Synthetic construct <400> 84 aacatcgtgc tgacccagtc cccagactct ctggccgtgt ccctgggaga gagggccaca 60 atcaactgca gaccctctga gagcgtggat agctacggca attccttcat gcactggtat 120 cagcagaagc ctggccagcc ccctaagctg ctgatctacc tggcctctaa tctggagagc 180 ggcgtgccag acaggttctc cggatctgga agcggaaccg acttcaccct gacaatcagc 240 tccctgcagg cagaggacgt ggccgtgtac tattgtcagc agaacaatga ggatccccgg 300 acctttggcg gcggcacaaa ggtggagatc aagcgt 336 <210> 85 <211> 324 <212> DNA <213> Mus musculus <400> 85 gacatccaga tgacacaatc ttcatcctcc ttttctgtat ctctaggaga cagagtcacc 60 attacttgca aggcaagtga ggacatatat aatcggttcg cctggtttca gcagaaacca 120 ggaaatgctc ctaggctctt aatatctgat gcaaccagtt tggaaactgg ggttccttca 180 agattcagtg gcagtggatc tggaaaggat tacactctca gcattaccag tcttcagact 240 gaagatgttg ctacttatta ctgtcaacag tattggagta ttccgtggac gttcggtgga 300 ggcaccaagc tggaaatcaa acgg 324 <210> 86 <211> 336 <212> DNA <213> Mus musculus <400> 86 gacattgtgc tgacacagtc tcctgcttcc ttagctgtat ctctggggca gaggggcacc 60 atctcatgca gggccagcca aagtgtcaat acatctgtct atagttatat acactggtac 120 cggcagaaac caggtcagcc acccaaactc ctcatcaagt atgcatccaa cctagaatct 180 ggggtccctg ccaggttcag tggcagtggg tctgggacag acttcaccct caacatccat 240 cctgtggagg gggaggattc cgcaacatac tactgtcaac acagttggga ttttccttac 300 acgttcggag gggggaccaa gctggaaata aaacgg 336 <210> 87 <211> 336 <212> DNA <213> Artificial Sequence <220> <223> Synthetic construct <400> 87 gacatcgtgc tgacccagtc tccagccagc ctggccgtgt ctccaggaca gagggccacc 60 atcacatgca gagccagcca gtccgtgaac acagccgtgt actcttatat ccactggtac 120 cagcagaagc ctggccagcc ccctaagctg ctgatcaagt atgccagcaa cctggagtcc 180 ggagtgcctg cacggttctc tggaagcgga tccggaaccg acttcaccct gacaatcaat 240 ccagtggagg ccaacgacgc cgccaattac tattgtcagc acagcttcga tttcccctac 300 acctttggcg gcggcacaaa ggtggagatc aagcgt 336 <210> 88 <211> 336 <212> DNA <213> Artificial Sequence <220> <223> Synthetic construct <400> 88 gacatcgtgc tgacccagtc ccctgattct ctggccgtgt ccctgggaga gagggcaacc 60 atcaactgca gagcctctca gagcgtgaat acaagcgtgt actcctatat ccactggtac 120 cagcagaagc caggccagcc ccctaagctg ctgatcaagt atgcctctaa cctggagagc 180 ggagtgccag accggttctc cggctctggc agcggcacag acttcaccct gacaatcagc 240 tccctgcagg cagaggacgt ggccgtgtac tattgtcagc actcttggga tttcccctac 300 acctttggcg gcggcacaaa ggtggagatc aagcgt 336 <210> 89 <211> 324 <212> DNA <213> Mus musculus <400> 89 gacatcaaga tgacccagtc tccatcttcc gtgtatgcat ctctaggaga gagagtcact 60 atcacttgca aggcgagtca ggacattaat agctatttaa gctggttcca gcagaaacca 120 gggaaatctc ctaagaccct gatctatcgt gcaaacagat tggtagatgg ggtcccatca 180 aggttcagtg gcagtggatc tgggcaagat tattctctca ccatcagcag cctggagtat 240 gaagatatgg gaatttatta ttgtctacag tatgatgagt ttccattcac gttcggctcg 300 gggacaaagt tggaaatgaa acgg 324 <210> 90 <211> 330 <212> DNA <213> Homo sapiens <400> 90 agctacgtgc tgacccagcc acctagcgcc tccggaacac caggccagag ggtggccatc 60 tcttgcagcg gctccaactc taatatcggc tccaacaccg tgcactggta ccagcagctg 120 ccaggagcag cacccaagct gctgatctat tccaacaatc agcggccttc tggcgtgcca 180 gacagattca gcggctccaa ctctggcaca agcgcctccc tggccatctc tcggctgcag 240 agcgaggacg aggccgatta ctattgtgcc gcctgggacg attctctgaa tggcgtggtg 300 tttggcggcg gcaccaaggt gacagtgctg 330 <210> 91 <211> 330 <212> DNA <213> Homo sapiens <400> 91 agctacgtgc tgacccagcc acctagcgcc tccggaacac caggccagag ggtggccatc 60 tcttgcagcg gctccaactc taatatcggc tccaacaccg tgcactggta tcagcagctg 120 ccaggagcag cacccaagct gctgatctat ggcaacaatg atcggccttc tggcgtgcca 180 gacagattca gcggctccaa ctctggcaca agcgcctccc tggccatctc tcggctgcag 240 agcgaggacg aggccgatta ctattgtgcc gcctgggacg attctctgaa tggcgtggtg 300 tttggcggcg gtaccaaggt gacagtgctg 330 <210> 92 <211> 363 <212> DNA <213> Mus musculus <400> 92 gaagtgaagc tggtggagtc tgggggaggc ttagtgaagc ctggagggtc cctgaaactc 60 tcctgtgcag cctctggatt cactttcagt agctatgcca tgtcttgggt tcgcctgact 120 ccagaaaaaa ggctggagtg ggtcgcatcc attagtagtg gtggtgccac ctactatcca 180 gacagtgtga agggccgatt caccttctcc agagataatg ccaggaacat cctgtacctg 240 caaatgaaca gtctgaggtc tgaggacacg gccatgtatt actgtacaag aggcgagggc 300 ggtagtagct acccggcctg gtttgctttc tggggccaag ggactctggt cactgtctct 360 gca 363 <210> 93 <211> 378 <212> DNA <213> Mus musculus <400> 93 gaagtgcagc tggtggagtc tgggggaggc ttagtgaagc ctggagggtc cctgaaactc 60 tcctgtgcag cctctggatt cactttcagt agctatgcca tgtcttgggt tcgccagtct 120 ccagagaaga ggctggagtg ggtcgcagaa attagtagtg gtggtagtta cacctactat 180 ccagacactg tgacgggccg attcaccatc tccagagaca atgccaagaa caccctgtac 240 ctggaaatga gcagtctgag gtctgaggac acggccatgt attactgtgt aagggataag 300 gcgactcgaa ctggcatggg atttttttac catactatgg actactgggg tcaaggaacc 360 tcagtcaccg tctcctca 378 <210> 94 <211> 378 <212> DNA <213> Artificial Sequence <220> <223> Synthetic construct <400> 94 gaggtgcagc tggtggagtc tggaggagga ctggtgaagc caggaggaag cctgaggctg 60 tcctgcgcag cctctggctt cacctttagc tcctacgcca tgagctgggt gaggcaggca 120 ccaggcaagg gactggagtg ggtggccgag atctctagcg gcggctccta cacctactat 180 cctgacaccg tgacaggccg gttcacaatc agcagagata acgccaagaa ttccctgtat 240 ctgcagatga actctctgcg ggccgaggac accgccgtgt actattgcgt gcgggataag 300 gccacccgca caggcatggg cttcttttac cacacaatgg actattgggg ccagggcacc 360 ctggtgacag tgtcctct 378 <210> 95 <211> 354 <212> DNA <213> Mus musculus <400> 95 caggttcaac tgcagcagtc tggagctgag ctgatgaagc ctggggcctc agtgaagata 60 tcctgcaagg ctactggcta cacattcagt aggtactgga tagagtggat aaagcagagg 120 cctggacatg gccttgagtg gattggagag attttacctg gaagtgatag tcctaactac 180 aatgagaagt tcaagggcaa ggccacattc actgcaaata catcctccaa cacagcctac 240 atgcaactca gcagcctgac atttgaggac tctgccgtct attactgtgc aagaacggta 300 gtagctacaa ggtttgctta ctggggccaa gggactctgg tcactgtctt tgca 354 <210> 96 <211> 357 <212> DNA <213> Mus musculus <400> 96 gatgtgcagc ttcaggagtc gggacctggc ctggtgaaac cttctcagtc tctgtccctc 60 acctgcactg tcactggcta ctcaatcacc agtgattatg cctggaactg gatccggcag 120 tttcctggaa acaaagtgga gtggatgggc tacataagct acagaggcat cgctacctat 180 aaaccatctc tcaaaagtcg aatctctatc actcgagaca catccaagaa ccagttcttc 240 ctgaacttta attctgtgac tactgaggac acagccacat attactgtgt aagaggggaa 300 tacggccccg gcaactttga cttctggggc caaggcacca ctctcacagt ctcctca 357 <210> 97 <211> 357 <212> DNA <213> Artificial Sequence <220> <223> Synthetic construct <400> 97 caggtgcagc tgcaggagtc cggaccagga ctggtgaagc caagccagac cctgtccctg 60 acctgcacag tgtccggcta ctctatcaca agcgattatg cctggaactg gatcaggcag 120 ccacctggca agggagtgga gtggatgggc tacatctcct atcgcggcat cgccacctac 180 aagccttccc tgaagtctcg gatcaccatc tctagagaca caagcaagaa ccagttctct 240 ctgaagctga gctccgtgac cgccgccgat acagccgtgt actattgcgt gcggggcgag 300 tatggccccg gcaatttcga cttttggggc cagggcacca cagtgacagt gtctagc 357 <210> 98 <211> 357 <212> DNA <213> Mus musculus <400> 98 gatgtgcagc ttcaagagtc gggacctggc ctggtgaaac cttctcagtc tctgtccctc 60 acctgcactg tcactggcta ctcaatcacc agtgattatg cctggaactg gatccggcag 120 tttccaggaa acaaactgga gtggatgggc tacatgagct accgtggtac cgcaacgtac 180 aatccatttc tcaaaagtcg aatctctatc aatcgggaca catccaagaa ccaggtcttc 240 ctgaagttga attctgtgac tactgaggac acagccacat attactgtgc aagttatgat 300 tacgacgttc cccggtttcc ttactggggc caagggactc tggtcattgt ttctgca 357 <210> 99 <211> 357 <212> DNA <213> Homo sapiens <400> 99 caggtgcagc tgcagcagag cggagcagag gtgaagaagc caggaagctc cgtgaaggtg 60 tcctgcaagg cctctggcgg caccttctct agctacgcca tctcctgggt gcggcaggca 120 ccaggacagg gactggagtg gatgggagga atcatcccca ccttcggcac agccaactac 180 gcccagaagt ttcagggccg ggtgaccatc acagccgacg agtctaccag cacagcctat 240 atggagctgt cctctctgag atctgaggat accgccgtgt actattgtgc acagggacca 300 atcgtgggag cacctacaga ctattggggc aagggcaccc tggtgacagt gagctcc 357 <210> 100 <211> 357 <212> DNA <213> Homo sapiens <400> 100 caggtgcagc tgcagcagag cggagcagag gtgaagaagc caggaagctc cgtgaaggtg 60 tcctgcaagg cctctggcgg caccttcaag agctacgcca tctcctgggt gcggcaggca 120 ccaggacagg gactggagtg gatgggagga atcatcccca ccttcggcac agccaactac 180 gcccagaagt ttcagggccg ggtgaccatc acagccgacg agtctaccag cacagcctat 240 atggagctgt cctctctgag atctgaggat accgccgtgt actattgtgc acagggacca 300 atcgtgggag cacctacaga ctattggggc aagggcaccc tggtgacagt gagctcc 357 <210> 101 <211> 106 <212> PRT <213> Homo sapiens <400> 101 Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln 1 5 10 15 Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr 20 25 30 Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser 35 40 45 Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr 50 55 60 Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys 65 70 75 80 His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro 85 90 95 Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 100 105 <210> 102 <211> 106 <212> PRT <213> Homo sapiens <400> 102 Gly Gln Pro Lys Ala Ala Pro Ser Val Thr Leu Phe Pro Pro Ser Ser 1 5 10 15 Glu Glu Leu Gln Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp 20 25 30 Phe Tyr Pro Gly Ala Val Thr Val Ala Trp Lys Ala Asp Ser Ser Pro 35 40 45 Val Lys Ala Gly Val Glu Thr Thr Thr Pro Ser Lys Gln Ser Asn Asn 50 55 60 Lys Tyr Ala Ala Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys 65 70 75 80 Ser His Arg Ser Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr Val 85 90 95 Glu Lys Thr Val Ala Pro Thr Glu Cys Ser 100 105 <210> 103 <211> 326 <212> PRT <213> Homo sapiens <220> <221> MISC_FEATURE <222> (131)..(131) <223> Xaa at position 131 is Met or Tyr <220> <221> MISC_FEATURE <222> (133)..(133) <223> Xaa at position 133 is Ser or Thr <220> <221> MISC_FEATURE <222> (135)..(135) <223> Xaa at position 135 is Thr or Glu <220> <221> MISC_FEATURE <222> (307)..(307) <223> Xaa at position 307 is Met or Leu <220> <221> MISC_FEATURE <222> (313)..(313) <223> Xaa at position 313 is Asn or Ser <220> <221> MISC_FEATURE <222> (326)..(326) <223> Xaa at position 326 is Lys or is absent <400> 103 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg 1 5 10 15 Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Asn Phe Gly Thr Gln Thr 65 70 75 80 Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Thr Val Glu Arg Lys Cys Cys Val Glu Cys Pro Pro Cys Pro Ala Pro 100 105 110 Pro Val Ala Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp 115 120 125 Thr Leu Xaa Ile Xaa Arg Xaa Pro Glu Val Thr Cys Val Val Val Asp 130 135 140 Val Ser His Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly 145 150 155 160 Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn 165 170 175 Ser Thr Phe Arg Val Val Ser Val Leu Thr Val Val His Gln Asp Trp 180 185 190 Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro 195 200 205 Ala Pro Ile Glu Lys Thr Ile Ser Lys Thr Lys Gly Gln Pro Arg Glu 210 215 220 Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn 225 230 235 240 Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile 245 250 255 Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr 260 265 270 Thr Pro Pro Met Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys 275 280 285 Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys 290 295 300 Ser Val Xaa His Glu Ala Leu His Xaa His Tyr Thr Gln Lys Ser Leu 305 310 315 320 Ser Leu Ser Pro Gly Xaa 325 <210> 104 <211> 327 <212> PRT <213> Homo sapiens <220> <221> MISC_FEATURE <222> (132)..(132) <223> Xaa at position 132 is Met or Tyr <220> <221> MISC_FEATURE <222> (134)..(134) <223> Xaa at position 134 is Ser or Thr <220> <221> MISC_FEATURE <222> (136)..(136) <223> Xaa at position 136 is Thr or Glu <220> <221> MISC_FEATURE <222> (308)..(308) <223> Xaa at position 308 is Met or Leu <220> <221> MISC_FEATURE <222> (314)..(314) <223> Xaa at position 314 is Asn or Ser <220> <221> MISC_FEATURE <222> (327)..(327) <223> Xaa at position 327 is Lys or is absent <400> 104 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg 1 5 10 15 Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Lys Thr 65 70 75 80 Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro Ser Cys Pro Ala Pro 100 105 110 Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys 115 120 125 Asp Thr Leu Xaa Ile Xaa Arg Xaa Pro Glu Val Thr Cys Val Val Val 130 135 140 Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp 145 150 155 160 Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe 165 170 175 Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp 180 185 190 Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu 195 200 205 Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg 210 215 220 Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys 225 230 235 240 Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp 245 250 255 Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys 260 265 270 Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser 275 280 285 Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser 290 295 300 Cys Ser Val Xaa His Glu Ala Leu His Xaa His Tyr Thr Gln Lys Ser 305 310 315 320 Leu Ser Leu Ser Leu Gly Xaa 325 <210> 105 <211> 29 <212> PRT <213> Homo sapiens <400> 105 His Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly 1 5 10 15 Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Gly 20 25 <210> 106 <211> 29 <212> PRT <213> Artificial Sequence <220> <223> Synthetic construct <400> 106 His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly 1 5 10 15 Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Lys Gly 20 25 <210> 107 <211> 22 <212> PRT <213> Artificial Sequence <220> <223> Synthetic construct <400> 107 His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly 1 5 10 15 Gln Ala Ala Lys Glu Phe 20 <210> 108 <211> 23 <212> PRT <213> Artificial Sequence <220> <223> Synthetic construct <400> 108 His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly 1 5 10 15 Gln Ala Ala Lys Glu Phe Ile 20 <210> 109 <211> 24 <212> PRT <213> Artificial Sequence <220> <223> Synthetic construct <400> 109 His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly 1 5 10 15 Gln Ala Ala Lys Glu Phe Ile Ala 20 <210> 110 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Synthetic construct <400> 110 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 1 5 10 <210> 111 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> Synthetic construct <400> 111 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 1 5 10 15 <210> 112 <211> 20 <212> PRT <213> Artificial Sequence <220> <223> Synthetic construct <400> 112 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly 1 5 10 15 Gly Gly Gly Ser 20 <210> 113 <211> 466 <212> PRT <213> Homo sapiens <400> 113 Met Thr Thr Ser Pro Ile Leu Gln Leu Leu Leu Gly Leu Ser Leu Cys 1 5 10 15 Gly Leu Leu Leu Gln Arg Ala Glu Thr Gly Ser Lys Gly Gln Thr Ala 20 25 30 Gly Glu Leu Tyr Gln Arg Trp Glu Arg Tyr Arg Arg Glu Cys Gln Glu 35 40 45 Thr Leu Ala Ala Ala Glu Pro Pro Ser Gly Leu Ala Cys Asn Gly Ser 50 55 60 Phe Asp Met Tyr Val Cys Trp Asp Tyr Ala Ala Pro Asn Ala Thr Ala 65 70 75 80 Arg Ala Ser Cys Pro Trp Tyr Leu Pro Trp His His His Val Ala Ala 85 90 95 Gly Phe Val Leu Arg Gln Cys Arg Ser Asp Gly Gln Trp Gly Leu Trp 100 105 110 Arg Asp His Thr Gln Cys Glu Asn Pro Glu Lys Asn Glu Ala Phe Leu 115 120 125 Asp Gln Arg Leu Ile Leu Glu Arg Leu Gln Val Met Tyr Thr Val Gly 130 135 140 Tyr Ser Leu Ser Leu Ala Thr Leu Leu Leu Ala Leu Leu Ile Leu Ser 145 150 155 160 Leu Phe Arg Arg Leu His Cys Thr Arg Asn Tyr Ile His Ile Asn Leu 165 170 175 Phe Thr Ser Phe Met Leu Arg Ala Ala Ala Ile Leu Ser Arg Asp Arg 180 185 190 Leu Leu Pro Arg Pro Gly Pro Tyr Leu Gly Asp Gln Ala Leu Ala Leu 195 200 205 Trp Asn Gln Ala Leu Ala Ala Cys Arg Thr Ala Gln Ile Val Thr Gln 210 215 220 Tyr Cys Val Gly Ala Asn Tyr Thr Trp Leu Leu Val Glu Gly Val Tyr 225 230 235 240 Leu His Ser Leu Leu Val Leu Val Gly Gly Ser Glu Glu Gly His Phe 245 250 255 Arg Tyr Tyr Leu Leu Leu Gly Trp Gly Ala Pro Ala Leu Phe Val Ile 260 265 270 Pro Trp Val Ile Val Arg Tyr Leu Tyr Glu Asn Thr Gln Cys Trp Glu 275 280 285 Arg Asn Glu Val Lys Ala Ile Trp Trp Ile Ile Arg Thr Pro Ile Leu 290 295 300 Met Thr Ile Leu Ile Asn Phe Leu Ile Phe Ile Arg Ile Leu Gly Ile 305 310 315 320 Leu Leu Ser Lys Leu Arg Thr Arg Gln Met Arg Cys Arg Asp Tyr Arg 325 330 335 Val Arg Leu Ala Arg Ser Thr Leu Thr Leu Val Pro Leu Leu Gly Val 340 345 350 His Glu Val Val Phe Ala Pro Val Thr Glu Glu Gln Ala Arg Gly Ala 355 360 365 Leu Arg Phe Ala Lys Leu Gly Phe Glu Ile Phe Leu Ser Ser Phe Gln 370 375 380 Gly Phe Leu Val Ser Val Leu Tyr Cys Phe Ile Asn Lys Glu Val Gln 385 390 395 400 Ser Glu Ile Arg Arg Gly Trp His His Cys Arg Leu Arg Arg Ser Leu 405 410 415 Gly Glu Glu Gln Arg Gln Leu Pro Glu Arg Ala Phe Arg Ala Leu Pro 420 425 430 Ser Gly Ser Gly Pro Gly Glu Val Pro Thr Ser Arg Gly Leu Ser Ser 435 440 445 Gly Thr Leu Pro Gly Pro Gly Asn Glu Ala Ser Arg Glu Leu Glu Ser 450 455 460 Tyr Cys 465 <210> 114 <211> 1401 <212> DNA <213> Homo sapiens <400> 114 atgactacct ctccgatcct gcagctgctg ctcgggctct cactgtgcgg gctgctgctc 60 cagagggcgg agacaggctc taaggggcag actgcggggg agctgtacca gcgctgggaa 120 cggtaccgca gggagtgcca ggagaccttg gcagccgcgg aaccgccttc aggcctcgcc 180 tgtaacgggt ccttcgatat gtacgtctgc tgggactatg ctgcacccaa tgccactgcc 240 cgtgcgtcct gcccctggta cctgccctgg caccaccatg tggctgcagg tttcgtcctc 300 cgccagtgtc ggagtgatgg ccaatgggga ctttggagag accatacaca atgtgagaac 360 ccagagaaga atgaggcctt tctggaccaa aggctcatct tggagcggtt gcaggtcatg 420 tacactgtcg gctactccct gtctctcgcc acactgctgc tagccctgct catcttgagt 480 ttgttcaggc ggctacattg cactagaaac tatatccaca tcaacctgtt cacgtctttc 540 atgctgcgag ctgcggccat tctcagccga gaccgtctgc tacctcgacc tggcccctac 600 cttggggacc aggcccttgc gctgtggaac caggccctcg ctgcctgccg cacggcccag 660 atcgtgaccc agtactgcgt gggtgccaac tacacgtggc tgctggtgga gggcgtctac 720 ctgcacagtc tcctggtgct cgtgggaggc tccgaggagg gccacttccg ctactacctg 780 ctcctcggct ggggggcccc cgcgcttttc gtcattccct gggtgatcgt caggtacctg 840 tacgagaaca cgcagtgctg ggagcgcaac gaagtcaagg ccatttggtg gattatacgg 900 acccccatcc tcatgaccat cttgattaat ttcctcattt ttatccgcat tcttggcatt 960 ctcctgtcca agctgaggac acggcaaatg cgctgccggg attaccgcgt gaggctggct 1020 cgctccacgc tgacgctggt gcccctgctg ggtgtccacg aggtggtgtt tgctcccgtg 1080 acagaggaac aggcccgggg cgccctgcgt ttcgccaagc tcggctttga gatcttcctc 1140 agctccttcc agggcttcct ggtcagcgtc ctctactgct tcatcaacaa ggaggtgcag 1200 tcggagatcc gccgtggctg gcaccactgc cgcctgcgcc gcagcctggg cgaggagcaa 1260 cgccagctcc cggagcgcgc cttccgggcc ctgccctccg gctccggccc gggcgaggtc 1320 cccaccagcc gcggcttgtc ctcggggacc ctcccagggc ctgggaatga ggccagccgg 1380 gagttggaaa gttactgcta g 1401 <210> 115 <211> 466 <212> PRT <213> Macaca mulatta <400> 115 Met Thr Thr Ser Pro Ile Leu Gln Leu Leu Leu Arg Leu Ser Leu Trp 1 5 10 15 Gly Leu Leu Leu Arg Arg Ala Glu Thr Gly Ser Glu Gly Gln Thr Ala 20 25 30 Gly Glu Leu Tyr Gln Arg Trp Glu Arg Tyr Arg Arg Glu Cys Gln Glu 35 40 45 Thr Leu Ala Thr Ala Glu Pro Pro Ser Gly Leu Ala Cys Asn Gly Ser 50 55 60 Phe Asp Met Tyr Val Cys Trp Asn Tyr Ala Ala Pro Asn Ala Thr Ala 65 70 75 80 Arg Ala Ser Cys Pro Trp Tyr Leu Pro Trp His His His Val Ala Ala 85 90 95 Gly Phe Val Leu Arg Gln Cys Gly Ser Asp Gly Gln Trp Gly Leu Trp 100 105 110 Arg Asp His Thr Gln Cys Glu Asn Pro Glu Lys Asn Glu Ala Phe Leu 115 120 125 Asp Gln Arg Leu Ile Leu Glu Arg Leu Gln Val Met Tyr Thr Val Gly 130 135 140 Tyr Ser Leu Ser Leu Ala Thr Leu Leu Leu Ala Leu Leu Ile Leu Ser 145 150 155 160 Leu Phe Arg Arg Leu His Cys Thr Arg Asn Tyr Ile His Ile Asn Leu 165 170 175 Phe Thr Ser Phe Thr Leu Arg Ala Ala Ala Ile Leu Ser Arg Asp Arg 180 185 190 Leu Leu Pro Arg Pro Gly Pro Tyr Leu Gly Asp Gln Ala Leu Val Leu 195 200 205 Trp Asn Gln Ala Leu Ala Ala Cys Arg Thr Ala Gln Ile Val Thr Gln 210 215 220 Tyr Cys Val Gly Ala Asn Tyr Thr Trp Leu Leu Val Glu Gly Val Tyr 225 230 235 240 Leu His Ser Leu Leu Val Ile Val Gly Gly Ser Glu Glu Gly His Phe 245 250 255 Arg Tyr Tyr Leu Leu Leu Gly Trp Gly Ala Pro Ala Leu Phe Val Ile 260 265 270 Pro Trp Val Ile Val Arg Tyr Leu Tyr Glu Asn Thr Gln Cys Trp Glu 275 280 285 Arg Asn Glu Val Lys Ala Ile Trp Trp Ile Ile Arg Thr Pro Ile Leu 290 295 300 Met Thr Ile Leu Ile Asn Phe Leu Ile Phe Ile Arg Ile Leu Gly Ile 305 310 315 320 Leu Leu Ser Lys Leu Arg Thr Arg Gln Met Arg Cys Arg Asp Tyr Arg 325 330 335 Leu Arg Leu Ala Arg Ser Thr Leu Thr Leu Val Pro Leu Leu Gly Val 340 345 350 His Glu Val Val Phe Ala Pro Val Thr Glu Glu Gln Ala Arg Gly Ala 355 360 365 Leu Arg Phe Ala Lys Leu Gly Phe Glu Ile Phe Leu Ser Ser Phe Gln 370 375 380 Gly Leu Leu Val Ser Val Leu Tyr Cys Phe Ile Asn Lys Glu Val Gln 385 390 395 400 Ser Glu Ile Arg Arg Gly Trp His His Cys Arg Leu Arg Arg Ser Leu 405 410 415 Gly Glu Glu Gln Arg Gln Leu Pro Glu Arg Ala Phe Arg Ala Leu Pro 420 425 430 Ser Gly Ser Gly Pro Gly Glu Val Pro Thr Gly Arg Gly Leu Ser Ser 435 440 445 Gly Thr Leu Pro Gly Pro Gly Asn Glu Ala Arg Arg Val Leu Glu Ser 450 455 460 Tyr Cys 465 <210> 116 <211> 1398 <212> DNA <213> Macaca mulatta <400> 116 atgactacct ctccgatcct gcagttgctg ttgcggctct cactgtgggg gctgctgctc 60 cggagggcgg agacaggctc tgaggggcag acggcggggg agctgtacca gcgctgggaa 120 cggtaccgca gggagtgcca ggagaccttg gcaaccgcgg aaccaccttc aggcctcgcc 180 tgtaacgggt ccttcgatat gtacgtctgc tggaactatg ctgcacccaa cgccactgcc 240 cgtgcctcct gcccctggta cctgccctgg caccaccacg tggctgcagg tttcgtcctc 300 cgccagtgtg gcagtgatgg ccagtgggga ctttggagag accacacaca atgtgagaac 360 ccagagaaga atgaggcctt tctggaccaa aggctcatct tggagcggct gcaggtcatg 420 tacaccgtcg gctactccct gtctctcgcc acactgctgc tagccctgct catcttgagc 480 ttgttcaggc gactacattg cactagaaac tatatccaca tcaacctgtt cacgtctttc 540 acactgcgag cggcggcgat tctcagccgg gaccgtctac tgcctcgacc tggcccctac 600 cttggggacc aggcccttgt gctgtggaac caggccctcg ctgcctgccg cacggcccag 660 atcgtgaccc agtactgcgt gggtgccaac tacacgtggc tgctggtgga gggcgtctac 720 ttgcacagtc tcctggtgat cgtgggaggc tccgaggagg gtcacttccg ctactacctg 780 ctcctcggct ggggggcccc cgcgcttttc gtcattccct gggtgatcgt caggtacctg 840 tacgagaaca cgcagtgctg ggagcgcaac gaagtcaagg ccatttggtg gattatacgg 900 acccccatcc tcatgaccat cttgattaat ttcctcattt ttatccgcat tcttggcatt 960 ctcctgtcca agctgaggac acggcaaatg cgctgccggg attaccggct gaggctggct 1020 cgctccacgc tgacgctggt gcccctgctg ggtgtccacg aggtggtgtt tgctcccgtg 1080 accgaggaac aggcccgggg cgccctgcgc ttcgccaagc tcggctttga gatcttcctc 1140 agctctttcc agggcttgct ggtcagcgtc ctctactgct tcatcaacaa ggaggtgcag 1200 tcggagatcc gccgtggctg gcaccactgc cgcctgcgcc gcagcctggg cgaggagcag 1260 cggcagctcc cggagcgcgc cttccgggcc ctgccctccg gctccggccc cggcgaggtc 1320 cccaccggcc gcggcttgtc ttcggggacc ctcccagggc ctgggaatga ggccagacgg 1380 gtgttggaaa gttactgc 1398 <210> 117 <211> 460 <212> PRT <213> Mus musculus <400> 117 Met Pro Leu Arg Leu Leu Leu Leu Leu Leu Trp Leu Trp Gly Leu Gln 1 5 10 15 Trp Ala Glu Thr Asp Ser Glu Gly Gln Thr Thr Thr Gly Glu Leu Tyr 20 25 30 Gln Arg Trp Glu His Tyr Gly Gln Glu Cys Gln Lys Met Leu Glu Thr 35 40 45 Thr Glu Pro Pro Ser Gly Leu Ala Cys Asn Gly Ser Phe Asp Met Tyr 50 55 60 Ala Cys Trp Asn Tyr Thr Ala Ala Asn Thr Thr Ala Arg Val Ser Cys 65 70 75 80 Pro Trp Tyr Leu Pro Trp Phe Arg Gln Val Ser Ala Gly Phe Val Phe 85 90 95 Arg Gln Cys Gly Ser Asp Gly Gln Trp Gly Ser Trp Arg Asp His Thr 100 105 110 Gln Cys Glu Asn Pro Glu Lys Asn Gly Ala Phe Gln Asp Gln Thr Leu 115 120 125 Ile Leu Glu Arg Leu Gln Ile Met Tyr Thr Val Gly Tyr Ser Leu Ser 130 135 140 Leu Thr Thr Leu Leu Leu Ala Leu Leu Ile Leu Ser Leu Phe Arg Arg 145 150 155 160 Leu His Cys Thr Arg Asn Tyr Ile His Met Asn Leu Phe Thr Ser Phe 165 170 175 Met Leu Arg Ala Ala Ala Ile Leu Thr Arg Asp Gln Leu Leu Pro Pro 180 185 190 Leu Gly Pro Tyr Thr Gly Asp Gln Ala Pro Thr Pro Trp Asn Gln Ala 195 200 205 Leu Ala Ala Cys Arg Thr Ala Gln Ile Met Thr Gln Tyr Cys Val Gly 210 215 220 Ala Asn Tyr Thr Trp Leu Leu Val Glu Gly Val Tyr Leu His His Leu 225 230 235 240 Leu Val Ile Val Gly Arg Ser Glu Lys Gly His Phe Arg Cys Tyr Leu 245 250 255 Leu Leu Gly Trp Gly Ala Pro Ala Leu Phe Val Ile Pro Trp Val Ile 260 265 270 Val Arg Tyr Leu Arg Glu Asn Thr Gln Cys Trp Glu Arg Asn Glu Val 275 280 285 Lys Ala Ile Trp Trp Ile Ile Arg Thr Pro Ile Leu Ile Thr Ile Leu 290 295 300 Ile Asn Phe Leu Ile Phe Ile Arg Ile Leu Gly Ile Leu Val Ser Lys 305 310 315 320 Leu Arg Thr Arg Gln Met Arg Cys Pro Asp Tyr Arg Leu Arg Leu Ala 325 330 335 Arg Ser Thr Leu Thr Leu Val Pro Leu Leu Gly Val His Glu Val Val 340 345 350 Phe Ala Pro Val Thr Glu Glu Gln Val Glu Gly Ser Leu Arg Phe Ala 355 360 365 Lys Leu Ala Phe Glu Ile Phe Leu Ser Ser Phe Gln Gly Phe Leu Val 370 375 380 Ser Val Leu Tyr Cys Phe Ile Asn Lys Glu Val Gln Ser Glu Ile Arg 385 390 395 400 Gln Gly Trp Arg His Arg Arg Leu Arg Leu Ser Leu Gln Glu Gln Arg 405 410 415 Pro Arg Pro His Gln Glu Leu Ala Pro Arg Ala Val Pro Leu Ser Ser 420 425 430 Ala Cys Arg Glu Ala Ala Val Gly Asn Ala Leu Pro Ser Gly Met Leu 435 440 445 His Val Pro Gly Asp Glu Val Leu Glu Ser Tyr Cys 450 455 460 <210> 118 <211> 1380 <212> DNA <213> Mus musculus <400> 118 atgcccctgc ggttgctgct tctgctgctg tggttgtggg gactccagtg ggcggagaca 60 gactctgagg ggcagaccac cacgggggag ctgtaccagc gctgggagca ctacggccag 120 gagtgccaaa agatgttgga gaccacagaa cctccctcag gcctggcctg taacggttcc 180 ttcgatatgt atgcctgctg gaactacacg gccgccaaca ccactgctcg ggtgtcttgc 240 ccctggtatc tgccctggtt ccgtcaggtg tctgcaggct ttgtcttccg ccagtgtggc 300 agtgatggcc agtggggatc ttggagagac cacactcagt gtgagaatcc agagaagaat 360 ggggcttttc aggaccagac gctgatcctg gagcgcctgc agatcatgta taccgtgggc 420 tactccctgt ccctgacgac tctgctgcta gccctactca tcttaagttt gttcaggcgg 480 ctgcactgca ctcgtaatta cattcacatg aacctgttca cgtctttcat gctgcgggca 540 gcagccatcc tcacccgaga tcagctgctg cctccactgg gtccctacac tggagaccag 600 gcccctaccc cgtggaacca ggccctagct gcctgccgca cggcccagat catgacccaa 660 tattgtgtgg gagccaatta cacctggcta ctggtggagg gtgtttatct gcaccatctg 720 ctggtgatcg tgggacgctc agaaaagggc cacttccgct gctacctgct tcttggctgg 780 ggggcccccg cgcttttcgt cattccctgg gtgatcgtca ggtacctgcg cgagaacaca 840 cagtgctggg agcgcaacga agtcaaagcc atttggtgga tcattcgcac tcccatccta 900 ataaccatct tgatcaattt cctcatcttc atccgcatcc ttggcatcct tgtttcaaag 960 ctgaggacac ggcagatgcg ctgccccgac taccgactaa ggctggctcg ctccacgctg 1020 acactggtgc ccctgctggg tgtccacgag gtggtgtttg cgcctgtgac ggaggaacag 1080 gttgaaggct ccctgcgctt cgccaaactg gcctttgaaa tcttcctaag ttccttccag 1140 ggtttcctgg tgagcgtgct ctactgcttc atcaacaaag aggtgcagtc ggagatccgc 1200 cagggttggc gccaccgccg cctgcgtctc agtcttcaag agcagcgtcc acgtccgcac 1260 caggaactcg ccccccgggc tgtgcctttg agctctgcgt gccgagaagc tgccgttggc 1320 aacgccttgc cctctgggat gctgcatgtg cctggggatg aggtcttgga aagttactgc 1380 <210> 119 <211> 29 <212> PRT <213> Homo sapiens <400> 119 Gly Lys Val Leu Trp Ala Ile Phe Glu Lys Ala Ala Gln Gly Glu Leu 1 5 10 15 Tyr Ser Ser Val Asp Ser Thr Phe Thr Gly Glu Ala His 20 25 <210> 120 <211> 29 <212> PRT <213> Artificial Sequence <220> <223> Synthetic construct <400> 120 Gly Lys Val Leu Trp Ala Ile Phe Glu Lys Ala Ala Gln Gly Glu Leu 1 5 10 15 Tyr Ser Ser Val Asp Ser Thr Phe Thr Gly Glu Gly His 20 25 <210> 121 <211> 22 <212> PRT <213> Artificial Sequence <220> <223> Synthetic construct <400> 121 Phe Glu Lys Ala Ala Gln Gly Glu Leu Tyr Ser Ser Val Asp Ser Thr 1 5 10 15 Phe Thr Gly Glu Gly His 20 <210> 122 <211> 23 <212> PRT <213> Artificial Sequence <220> <223> Synthetic construct <400> 122 Ile Phe Glu Lys Ala Ala Gln Gly Glu Leu Tyr Ser Ser Val Asp Ser 1 5 10 15 Thr Phe Thr Gly Glu Gly His 20 <210> 123 <211> 24 <212> PRT <213> Artificial Sequence <220> <223> Synthetic construct <400> 123 Ala Ile Phe Glu Lys Ala Ala Gln Gly Glu Leu Tyr Ser Ser Val Asp 1 5 10 15 Ser Thr Phe Thr Gly Glu Gly His 20 <210> 124 <211> 326 <212> PRT <213> Homo sapiens <220> <221> MISC_FEATURE <222> (132)..(132) <223> Xaa at position 132 is Met or Tyr <220> <221> MISC_FEATURE <222> (134)..(134) <223> Xaa at position 134 is Ser or Thr <220> <221> MISC_FEATURE <222> (136)..(136) <223> Xaa at position 136 is Thr or Glu <220> <221> MISC_FEATURE <222> (308)..(308) <223> Xaa at position 308 is Met or Leu <220> <221> MISC_FEATURE <222> (314)..(314) <223> Xaa at position 314 is Asn or Ser <220> <221> MISC_FEATURE <222> (327)..(327) <223> Xaa at position 327 is Lys or is absent <400> 124 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg 1 5 10 15 Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Lys Thr 65 70 75 80 Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro 100 105 110 Glu Ala Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys 115 120 125 Asp Thr Leu Xaa Ile Xaa Arg Xaa Pro Glu Val Thr Cys Val Val Val 130 135 140 Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp 145 150 155 160 Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe 165 170 175 Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp 180 185 190 Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu 195 200 205 Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg 210 215 220 Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys 225 230 235 240 Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp 245 250 255 Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys 260 265 270 Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser 275 280 285 Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser 290 295 300 Cys Ser Val Xaa His Glu Ala Leu His Xaa His Tyr Thr Gln Lys Ser 305 310 315 320 Leu Ser Leu Ser Leu Gly Xaa 325

Claims (39)

An antibody that specifically binds to human GIPR, comprising a sequence of 1, 2, 3, 4, 5 or 6 amino acids, each amino acid sequence being independently selected from the amino acid sequences listed below. :
a. Light chain CDR1 amino acid sequence: SEQ ID NO: 1, SEQ ID NO: 4, SEQ ID NO: 7, SEQ ID NO: 10, SEQ ID NO: 13, and SEQ ID NO: 15;
b. Light chain CDR2 amino acid sequence: SEQ ID NO: 2, SEQ ID NO: 5, SEQ ID NO: 8, SEQ ID NO: 11, and SEQ ID NO: 16;
c. Light chain CDR3 amino acid sequence: SEQ ID NO: 3, SEQ ID NO: 6, SEQ ID NO: 9, SEQ ID NO: 12, SEQ ID NO: 14, and SEQ ID NO: 17;
d. Heavy chain CDR1 amino acid sequence: SEQ ID NO: 18, SEQ ID NO: 23, and SEQ ID NO: 26;
e. Heavy chain CDR2 amino acid sequence: SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 24, SEQ ID NO: 27, and SEQ ID NO: 29; And
f. Heavy chain CDR3 amino acid sequence: SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 25, SEQ ID NO: 28, and SEQ ID NO: 30. The antibody of claim 1, wherein the antibody comprises one or two amino acid sequences, and each amino acid sequence is independently selected from the amino acid sequences listed below:
a. Light chain CDR1 amino acid sequence: SEQ ID NO: 1, SEQ ID NO: 4, SEQ ID NO: 7, SEQ ID NO: 10, SEQ ID NO: 13, and SEQ ID NO: 15; And
b. Heavy chain CDR1 amino acid sequence: SEQ ID NO: 18, SEQ ID NO: 23, and SEQ ID NO: 26. The antibody of claim 1 or 2, wherein the antibody comprises one or two amino acid sequences, and each amino acid sequence is independently selected from the amino acid sequences listed below:
a. Light chain CDR2 amino acid sequence: SEQ ID NO: 2, SEQ ID NO: 5, SEQ ID NO: 8, SEQ ID NO: 11, and SEQ ID NO: 16; And
b. Heavy chain CDR2 amino acid sequence: SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 24, SEQ ID NO: 27, and SEQ ID NO: 29. The antibody of any one of claims 1 to 3, wherein the antibody comprises or further comprises one or two amino acid sequences, each amino acid sequence being independently selected from the amino acid sequences listed below:
a. Light chain CDR3 amino acid sequence: SEQ ID NO: 3, SEQ ID NO: 6, SEQ ID NO: 9, SEQ ID NO: 12, SEQ ID NO: 14, and SEQ ID NO: 17; And
b. Heavy chain CDR3 amino acid sequence: SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 25, SEQ ID NO: 28, and SEQ ID NO: 30. The antibody of any one of claims 1 to 4, wherein the antibody comprises or further comprises one or two amino acid sequences, each amino acid sequence being independently selected from the amino acid sequences listed below: SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO : 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, and SEQ ID NO: 17. The antibody of any one of claims 1 to 5, wherein the antibody comprises or further comprises one or two amino acid sequences, each amino acid sequence being independently selected from the amino acid sequences listed below: SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO : 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, and SEQ ID NO: 30. The antibody of any one of claims 1 to 6, wherein the antibody comprises or further comprises a combination of light and heavy chain CDR1 amino acid sequences independently selected from the list below: SEQ ID NO: 1 and SEQ ID NO: 18, SEQ ID NO: 4 and SEQ ID NO: 18, SEQ ID NO: 7 and SEQ ID NO: 23, SEQ ID NO: 10 and SEQ ID NO: 26, SEQ ID NO: 13 and SEQ ID NO: 26, and SEQ ID NO: 15 and SEQ ID NO: 26. The antibody of any one of claims 1 to 7, wherein the antibody comprises or further comprises a combination of light and heavy chain CDR2 amino acid sequences independently selected from the following list: SEQ ID NO: 2 and SEQ ID NO: 19, SEQ ID NO: 5 and SEQ ID NO: 21, SEQ ID NO: 8 and SEQ ID NO: 24, SEQ ID NO: 11 and SEQ ID NO: 27, and SEQ ID NO: 16 and SEQ ID NO : 29. The antibody of any one of claims 1 to 8, wherein the antibody comprises or further comprises a combination of light and heavy chain CDR3 amino acid sequences independently selected from the following list: SEQ ID NO: 3 and SEQ ID NO: 20, SEQ ID NO: 6 and SEQ ID NO: 22, SEQ ID NO: 9 and SEQ ID NO: 25, SEQ ID NO: 12 and SEQ ID NO: 28, SEQ ID NO: 14 and SEQ ID NO: 28, and SEQ ID NO: 17 and SEQ ID NO: 30. The antibody of any one of claims 1 to 9, wherein the antibody comprises one or two amino acid sequences, each amino acid sequence being independently selected from the amino acid sequences listed below:
a. Light chain variable domain sequence: SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO : 68, SEQ ID NO: 69, SEQ ID NO: 70, and SEQ ID NO: 71; And an amino acid sequence that is at least 80%, at least 85%, at least 90%, or at least 95% identical to any of the above sequences; And
b. Heavy chain variable domain sequence: SEQ ID NO: 72, SEQ ID NO: 73, SEQ ID NO: 74, SEQ ID NO: 75, SEQ ID NO: 76, SEQ ID NO: 77, SEQ ID NO: 78, SEQ ID NO : 79, and SEQ ID NO: 80; And an amino acid sequence that is at least 80%, at least 85%, at least 90%, or at least 95% identical to any of the above sequences. The antibody of any one of claims 1 to 10, wherein the polynucleotide encoding sequence comprises one or two polynucleotide sequences, and each polynucleotide sequence is independently selected from the polynucleotide sequences listed below. :
a. Light chain variable domain polynucleotide encoding sequence: SEQ ID NO: 81, SEQ ID NO: 82, SEQ ID NO: 83, SEQ ID NO: 84, SEQ ID NO: 85, SEQ ID NO: 86, SEQ ID NO: 87, SEQ ID NO: 88, SEQ ID NO: 89, SEQ ID NO: 90, and SEQ ID NO: 91; And a polynucleotide sequence that is at least 80%, at least 85%, at least 90%, or at least 95% identical to any of the above sequences; And
b. Heavy chain variable domain polynucleotide encoding sequence: SEQ ID NO: 92, SEQ ID NO: 93, SEQ ID NO: 94, SEQ ID NO: 95, SEQ ID NO: 96, SEQ ID NO: 97, SEQ ID NO: 98, SEQ ID NO: 99, and SEQ ID NO: 100; And a polynucleotide sequence that is at least 80%, at least 85%, at least 90%, or at least 95% identical to any of the above sequences. The antibody of any one of claims 1 to 11, wherein the antibody comprises or further comprises an amino acid sequence independently selected from the following list: SEQ ID NO: 61, SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID NO: 70, and SEQ ID NO : 71. The antibody of any one of claims 1-12, wherein the antibody comprises or further comprises an amino acid sequence independently selected from the following list: SEQ ID NO: 72, SEQ ID NO: 73, SEQ ID NO: 74, SEQ ID NO: 75, SEQ ID NO: 76, SEQ ID NO: 77, SEQ ID NO: 78, SEQ ID NO: 79, and SEQ ID NO: 80. The antibody of any one of claims 1-3, wherein the antibody comprises a combination of amino acid sequences independently selected from the following list: SEQ ID NO: 61 and SEQ ID NO: 72, SEQ ID NO: 62 and SEQ ID NO: 73, SEQ ID NO: 63 and SEQ ID NO: 74, SEQ ID NO: 64 and SEQ ID NO: 74, SEQ ID NO: 65 and SEQ ID NO: 75, SEQ ID NO: 66 and SEQ ID NO: 76, SEQ ID NO: 67 and SEQ ID NO: 77, SEQ ID NO: 68 and SEQ ID NO: 77, SEQ ID NO: 69 and SEQ ID NO: 78, SEQ ID NO: 70 and SEQ ID NO: 79, and SEQ ID NO: 71 and SEQ ID NO: 80. The antibody of any one of claims 1 to 14, wherein the antibody comprises or further comprises an amino acid sequence independently selected from the following list: SEQ ID NO: 62, SEQ ID NO: 63, SEQ ID NO: 64, SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 73, SEQ ID NO: 74, SEQ ID NO: 76, and SEQ ID NO: 77. The antibody of any one of claims 1 to 15, wherein the antibody comprises a combination of amino acid sequences independently selected from the following list: SEQ ID NO: 62 and SEQ ID NO: 73, SEQ ID NO: 63 and SEQ ID NO: 74, SEQ ID NO: 64 and SEQ ID NO: 74, SEQ ID NO: 66 and SEQ ID NO: 76, SEQ ID NO: 67 and SEQ ID NO: 77, and SEQ ID NO: 68 And SEQ ID NO: 77. The antibody of any one of claims 1 to 16, wherein the antibody comprises one or two amino acid sequences, each amino acid sequence being independently selected from the amino acid sequences listed below:
a. Light chain constant amino acid sequence: SEQ ID NO: 101 and SEQ ID NO: 102; And
b. Heavy chain constant amino acid sequence: SEQ ID NO: 103, SEQ ID NO: 104 and SEQ ID NO: 124. The antibody of any one of claims 1 to 17, wherein the antibody is a murine GIPR antibody or a humanized GIPR antibody. 19. The antibody of any one of claims 1-18, wherein the antibody is a GIPR monoclonal antibody. The antibody of any one of claims 1 to 19, wherein the antibody is a monoclonal antibody comprising a combination of amino acid sequences independently selected from the following list: SEQ ID NO: 61 and SEQ ID NO: 72 , SEQ ID NO: 62 and SEQ ID NO: 73, SEQ ID NO: 63 and SEQ ID NO: 74, SEQ ID NO: 64 and SEQ ID NO: 74, SEQ ID NO: 65 and SEQ ID NO: 75, SEQ ID NO: 66 and SEQ ID NO: 76, SEQ ID NO: 67 and SEQ ID NO: 77, SEQ ID NO: 68 and SEQ ID NO: 77, SEQ ID NO: 69 and SEQ ID NO: 78, SEQ ID NO : 70 and SEQ ID NO: 79, and SEQ ID NO: 71 and SEQ ID NO: 80. The method of any one of claims 1 to 20, wherein the antibody is a murine antibody, human antibody, humanized antibody, chimeric antibody, monoclonal antibody, polyclonal antibody, recombinant antibody, antigen-binding antibody fragment, single chain. From antibody, double chain antibody, triple chain antibody, quad chain antibody, Fab fragment, F(ab') _x fragment, domain antibody, IgD antibody, IgE antibody, IgM antibody, IgG1 antibody, IgG2 antibody, IgG3 antibody, or IgG4 antibody Selected, antibody. 22. The antibody of any one of claims 1-21, wherein the antibody has an IC50 of approximately 1 nM to 200 nM or 1 nM to 100 nM in reducing signal transduction of human GIP. A GLP-1 fusion protein, wherein the fusion protein is the GIPR antibody provided in claims 1 to 22, and 1, 2, 3, 4, 5, 6, 7 or 8 GLP-1 Structurally characterized as comprising fragments or reverse GLP-1 fragments; The fusion protein connects the carboxy end of the GLP-1 fragment with the amino end of the light or heavy chain of the GIPR antibody through a peptide linker, or connects the amino end of the reverse GLP-1 fragment with the carboxy end of the light or heavy chain of the GIPR antibody. A fusion protein, characterized in that it is structural. 24. The method of claim 23, wherein the fusion protein comprises a GIPR antibody and 1, 2, 3 or 4 GLP-1 fragments; The fusion protein is a fusion protein that connects the carboxy terminus of the GLP-1 fragment to the amino terminus of the light or heavy chain of the GIPR antibody through a peptide linker. 24. The method of claim 23, wherein the fusion protein comprises a GIPR antibody and one, two, three or four reverse GLP-1 fragments; The fusion protein is a fusion protein that connects the amino terminal of the reverse GLP-1 fragment with the carboxy terminal of the light or heavy chain of the GIPR antibody through a peptide linker. 24. The method of claim 23, wherein the fusion protein comprises a GIPR antibody and two GLP-1 fragments; The fusion protein is a fusion protein that connects the carboxy terminus of the GLP-1 fragment to the amino terminus of the light or heavy chain of the GIPR antibody through a peptide linker. 24. The method of claim 23, wherein the fusion protein comprises a GIPR antibody and two reverse GLP-1 fragments; The fusion protein is a fusion protein that connects the amino terminal of the reverse GLP-1 fragment with the carboxy terminal of the light or heavy chain of the GIPR antibody through a peptide linker. The fusion protein of claim 23, wherein the GIPR antibody, GLP-1 fragment and peptide linker are fused to form the fusion protein in one of the following manners:
Through a peptide linker, the carboxy terminus of the GLP-1 fragment is fused to the amino terminus of the light chain of the GIPR antibody: N'-GLP-1-Linker-R-C';
Through a peptide linker, the carboxy terminus of the GLP-1 fragment is fused to the amino terminus of the heavy chain of the GIPR antibody: N'-GLP-1-Linker-R-C';
(Wherein, N'represents the amino terminal of the fusion protein polypeptide chain, C'represents the carboxy terminal of the fusion protein polypeptide chain, GLP-1 represents the GLP-1 fragment, and R represents the term 1 to Represents the amino acid sequence of the light chain or heavy chain of the GIPR antibody of claim 22, and Linker represents a polypeptide linker). The method of any one of claims 23 to 28, wherein the peptide linker comprises a full length, partial, or repeating amino acid sequence independently selected from SEQ ID NO: 110, SEQ ID NO: 111, and SEQ ID NO: 112. Containing, GLP-1 fusion protein. The method of any one of claims 23 to 29, wherein the GLP-1 fragment is SEQ ID NO: 105, SEQ ID NO: 106, SEQ ID NO: 107, SEQ ID NO: 108, and SEQ ID NO: 109 Comprises an amino acid sequence independently selected from; The reverse GLP-1 fragment comprises an amino acid sequence independently selected from SEQ ID NO: 119, SEQ ID NO: 120, SEQ ID NO: 121, SEQ ID NO: 122, and SEQ ID NO: 123, GLP- 1 fusion protein. A polynucleotide encoding the GIPR antibody of any one of claims 1 to 22, or the GLP-1 fusion protein of any one of claims 23 to 30. A vector comprising the polynucleotide of claim 31. A host cell comprising the vector of claim 32. A pharmaceutical composition comprising the GIPR antibody of any one of claims 1 to 22 or the GLP-1 fusion protein of any one of claims 23 to 30, mixed with a pharmaceutically acceptable carrier. A pharmaceutical composition comprising the GIPR antibody of any one of claims 1 to 22 or the GLP-1 fusion protein of any of claims 23 to 29 in the manufacture of a medicament for preventing or treating non-alcoholic fatty liver disease. Use of. A pharmaceutical composition comprising the GIPR antibody of any one of claims 1 to 22 or the GLP-1 fusion protein of any of claims 23 to 29 in the manufacture of a medicament for preventing or treating type 2 diabetes. Use of. Comprising the GIPR antibody of any one of claims 1 to 22 or the GLP-1 fusion protein of any one of claims 23 to 29 in the manufacture of a medicament for preventing or treating obesity and obesity-related diseases. Use of pharmaceutical compositions. In the manufacture of a medicament for simultaneously preventing or treating two or more diseases of non-alcoholic fatty liver disease, obesity, or type 2 diabetes, the GIPR antibody of any one of claims 1 to 22 or any of claims 23 to 29 Use of a pharmaceutical composition comprising the GLP-1 fusion protein of claim 1. 39. Use according to any one of claims 35 to 38, wherein the pharmaceutical composition is administered intravenously or subcutaneously.

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